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Fakultät für Biologie und Biotechnologie
Lehrstuhl für Biologie der Mikroorganismen
Struktur und Funktion neuartiger RNA-Thermometer
Dissertation zur Erlangung des Gradeseines Doktors der Naturwissenschaften
der Fakultät für Biologie und Biotechnologieder Ruhr-Universität Bochum
angefertigt amLehrstuhl für Biologie der Mikroorganismen
vorgelegt vonJens Frank Kortmann
ausHagen
Referent: Prof. Dr. Franz NarberhausKorreferent: Prof. Dr. Ulrich Kück
Bochum 2010
Structure and function of novel RNA thermometers
Dissertation
Jens Frank Kortmann
Bochum 2010
Fakultät für Biologie und Biotechnologie
Lehrstuhl für Biologie der Mikroorganismen
Structure and function of novel RNA thermometers
Dissertation zur Erlangung des Gradeseines Doktors der Naturwissenschaften
der Fakultät für Biologie und Biotechnologieder Ruhr-Universität Bochum
angefertigt amLehrstuhl für Biologie der Mikroorganismen
vorgelegt vonJens Frank Kortmann
ausHagen
Referent: Prof. Dr. Franz NarberhausKorreferent: Prof. Dr. Ulrich Kück
Bochum 2010
Danksagung
Ich möchte meinem Doktorvater Herrn Prof. Dr. Franz Narberhaus für den großen inhaltlichen Freiraum danken, der mir bei der Wahl meiner Forschungsthemen gelassen wurde. Vielen Dank für die fortwährende Unterstützung, die Ermutigungen und das Vertrauen in den schwierigen Phasen meiner Arbeit. Eigeninitiative wurde immer gefördert und, wo notwendig, in die richtigen Bahnen gelenkt. Vielen Dank für die zahlreichen guten Ideen, die Hinweise zu deren pragmatischer Umsetzung und vor allem für die schöne Promotionszeit.
Herrn Prof. Dr. Ulrich Kück danke ich für die freundliche Übernahme des Zweitgutachtens und das fortwährend aufrichtige Interesse an meiner Arbeit.
Ich möchte mich bei Herrn Dr. Bernd Masepohl für seine wissenschaftliche Neugierde und für viele gute Hinweise und spannende Anekdoten aus der Experimentatoren-Welt bedanken. Bernds Tür steht jedem offen.
Frau Prof. Dr. Nicole Frankenberg-Dinkel danke ich für die vielen guten Anregungen und Vorschläge und die tolle wissenschaftliche Unterstützung während meiner Arbeit.
Ich danke Herrn Prof. Dr. Karl E. Klose für seinen Besuch in Deutschland, die unzähligen hilfreichen Mails und für eine rundum tolle Kooperation.
Frau Prof. Dr. Petra Dersch und Frau Katja Böhme danke ich für zahlreiche inspirierende Diskussionen an diversen Posterwänden und für die hervorragende Zusammenarbeit.
Ich danke der Studienstiftung des Deutschen Volkes für die finanzielle, vor allem aber für die exzellente ideelle Unterstützung während meiner Doktorarbeit. Dieser Dank gilt auch der Ruhr-University Research School, die ich ein Jahr lang aktiv mitgestalten durfte.
Ich möchte meinen Mitarbeitern aus der RNA-Subgruppe danken. Vor allem Frau Ulla Aschke für ihre tatkräftige Unterstützung im Labor und für ihre beinahe unheimliche Fähigkeit, wirklich „alles überall rein“ zu klonieren. Meinen Diplomanden Simon Sczodrok und Annika Cimdins danke ich für eine spannende Betreuungszeit, in der auch ich viel gelernt habe. Frau Dr. Birgit Klinkert danke ich für die Unterstützung und stete Hilfe bei allen Problemen. Ich danke den „kleinen RNAs“ für nützliche Tipps und prompte Unterstützung. Vielen Dank an Frau Rosemarie Gurski für die gemeinsame Bewältigung der Übungen in Prokaryontengenetik und die gute Hilfe beim Strahlenschutz. Danke für die gute Zeit, danke an sämtliche RNA-Mitstreiter für die Hilfe bei der Fertigstellung dieser Arbeit und das Aufrechterhalten meiner Vitalfunktionen in der Endphase dieser Arbeit.
Vielen Dank an Frau Petra Krämer für die schnelle Hilfe bei allen verwaltungstechnischen Problemen und die ununterbrochene Versorgung mit Koffein und kurzkettigen Kohlenhydraten.
Vielen Dank an den Lehrstuhl für Biochemie der Pflanzen für das Bereitstellen von Laborraum und die experimentellen Hilfestellungen.
Ich möchte sämtlichen Arbeitsgruppen am Lehrstuhl für Biologie der Mikroorganismen für die Hilfsbereitschaft in allen Forschungs-Situationen danken. Danke für das gute Arbeitsklima und die angenehme Zusammenarbeit.
Ich danke all meinen Freunden für Ihr Verständnis und die riesige moralische Unterstützung in den letzten Jahren. Bei Herrn Dr. Matthias Heyden möchte ich mich für die gemeinsame Zeit, die fortwährende Unterstützung und Freundschaft seit Wohnheimstagen bedanken. Herrn Dr. Sebastian Rasche danke ich für seine wissenschaftliche Hilfe und den Rock N‘ Roll. Bei Herrn Dr. Mathias Hentrich und Herrn Björn Moll möchte ich mich für viel Inspiration und eine wirklich gute Zeit bedanken.
Herrn Ernst Skerra danke ich für das vermittelte Wissen und seine jahrelange Freundschaft.
Bei Jaclyn Dalisay möchte ich mich für die unentwegte Unterstützung in den letzten Jahren bedanken. Dieser Dank gilt auch ihrer Familie.
Ich danke meiner Familie, vor allem meinen Eltern und meinem kleinen Bruder für die Unterstützung in der langen Promotionszeit. Ihr habt sehr viel zum guten Gelingen dieser Arbeit beigetragen. Danke.
- It ain‘t over till it‘s over. -- Never give up. And never stop believing. -
Robert „Rocky“ Balboa
I. Table of contents
I Table of contents I
II Abbreviations II
A Introduction 1 1. Micros for microbes - small RNAs regulate bacterial gene expression 1 2. The cis-active case - riboswitches and RNA thermometers 2 3. The E. coli rpoH thermometer 4 4. ROSE - Repression Of heat Shock gene Expression 5 5. Cold shock thermosensors 7 6. RNA thermometers in bacterial pathogens 7
B Objectives 12
C Generation of synthetic RNA-based thermosensors 13
D Translation on demand by a simple RNA-based thermosensor 14
E Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader is based on nucleotide slippage and pseudoknot formation 15
F Discussion 16 1. Synthetic biology - molecular tailoring of biological systems at the nanoscale 16 2. RNA synthetic biology inspired by microbes: construction of translation initiators under temperature control 16 3. Potential applications of the synthetic RNA thermometer module 19 4. What it takes to be an RNA thermometer - the consensus algorithm 21 5. Destabilization of a bipartite stem - a novel principle among cyanobacterial RNA thermometers? 23 6. Cyotherm - a novel family of „green“ RNA thermometers 24 7. High light-sensing by a heat shock RNA thermometer? How co-translational protein insertion might contribute to it 26 8. RNA elements as antibacterial drug targets? 28 9. Anti-thermometer drugs - a promising attempt? 31
G Summary 33
I. Table of contents
H Zusammenfassung 35
I References 37
J Conference contributions 45 1. Posters 45 2. Talks 46
K Appendix 47 1. Curriculum vitae 47 2. Erklärung 48
L Eigenanteil an den Manuskripten 49
I. Table of contents
II. Abbreviations
AUG translational start site (methionine codon)
CD circular dichroism
dNTP desoxynucleotide triphosphate
Fig. figure
mRNA messenger RNA
MU Miller Units
kDa kilodalton
NTP nucleoside triphosphate
ORF open reading frame
RBS ribosome binding site
RNAP RNA polymerase
ROSE Repression Of heat Shock gene Expression
rRNA ribosomal RNA
SD Shine Dalgarno
UTR untranslated region
II. Abbreviations
A. Introduction
In order to survive in an often harsh environment, a microbe has to constantly monitor changes in
environmental conditions and adjust its metabolism appropriately. Such fine-tuning of gene-
expression is especially important for pathogenic bacteria, which need to employ several protective
measures during host-infection. For many years, these mechanisms were thought to be regulated
exclusively by proteins at the transcriptional level. Today there is an extensive body of literature
that describes the importance of post-transcriptional regulation in both eukaryotic and prokaryotic
microorganisms. It has become clear that mRNA is not a passive carrier of genetic information.
Instead it can form three-dimensional structures and thereby control its ability to initiate translation
or being degraded. Recent data reveals RNA as an ubiquitous mediator of gene expression.
1. Micros for microbes - small RNAs regulate bacterial gene expression
Regulatory RNAs can roughly be separated into two groups. The trans-acting small RNAs (sRNAs)
function by base-pairing with their target mRNA encoded elsewhere on the chromosome, and
thereby control its fate (1). A noteworthy subclass are antisense sRNAs (asRNAs) that are
encoded on the DNA strand opposite to its target mRNA and share perfect complementarity with
their target (2,3). In contrast, other transcripts contain cis-active built-in elements that directly affect
expression of the downstream coding sequence. The majority of the regulation by sRNA is
negative as base pairing between the sRNA and its target mostly results in translational inhibition,
mRNA degradation or both (4-6). Many sRNAs occlude the SD-harbouring ribosome binding site
(RBS), although some sRNAs like GcvB and RyhB, regulate their targets by base pairing far
upstream of the RBS (7,8). Having an average size around 100 nucleotides (although longer
variants over 500 nucleotides are known, (9)) bacterial sRNAs are larger than their eukaryotic
counterparts (around 25 nucleotides longer). As bacterial sRNAs provide different regions of
complementarity, one sRNA is often capable to control several mRNA targets in a synchronized
manner. The above-mentioned Escherichia coli sRNA RyhB binds many of the iron-storage
encoding mRNAs and thereby initiates their decay (7). RyhB is absent under high iron
concentrations which allows expression of iron-storage proteins.
A. Introduction
1
In many cases, the hexameric RNA chaperone Hfq is necessary for sRNA-mediated
regulation, presumably by facilitating RNA-RNA interactions despite limited complementarity
between the sRNA and the respective target mRNA (10,11).
2. The cis-active case - riboswitches and RNA thermometers
The first described class of cis-regulatory elements were classic attenuators that reside in the 5‘-
end of bacterial mRNAs. During transcription, stalled ribosomes lead to changes in the mRNA
structure, affecting transcription elongation through the formation of terminator or anti-terminator
structures in the mRNA (12, 13). A related mechanism is widely used in gram-positive bacteria
where sequences found in transcripts encoding aminoacyl-tRNA-synthetases, termed „T-boxes“,
bind the corresponding uncharged tRNA. This stabilizes the anti-terminator structure in preference
to the terminator structure, thereby preventing transcription termination (14, 15).
More recently, it was found that structured 5‘-untranslated regions (UTRs) of certain
bacterial mRNAs, riboswitches, bind specific metabolites and adopt different conformations in the
presence or absence of these small molecules (16-18). As the genes regulated by a riboswitch are
usually involved in the synthesis or transport of its specific metabolite, riboswitches are direct
modulators of cellular metabolite concentrations (19). If the cognate metabolite is not available
during transcription of the 5‘-UTR („GENE ON“, Fig.1a, b), the riboswitch in most cases folds into a
structure that does not interfere with the expression of the adjacent open reading frame (ORF). If
the concentration rises to a sufficiently high level, the metabolite binds to the riboswitch receptor
domain, which initiates formation of a structure in the nascent transcript that prevents the
expression of the ORF. This structure can either be a terminator, which stops RNA synthesis
prematurely („GENE OFF“, Fig. 1a) or a hairpin that masks the SD site and prevents the ribosome
from binding to the mRNA and translating the ORF („GENE OFF“, Fig. 1b).
A. Introduction
2
At least 20 different classes of riboswitches specific to different metabolites are known (20).
This suggests that the diversity of riboswitch function and/or control mechanisms could be greater
than envisioned originally. In 2009, Shoshy Altuvia (Jerusalem, Israel) presented the first example
of a pH-responsive riboswitch introducing a novel concept that involves folding dynamics driven by
pH (21). The pH-responsive RNA element resides upstream of E. coli alx, a gene encoding an
A
B
C
RNAP
ORF
ORF
ORF
ORF
ORF
- Ligand + Ligand
mRNA 5‘
5ʻ
mRNA 5‘
5‘ 5‘
5‘
5‘
GENE ON GENE OFF
Anti-Terminator
Terminator
UUUUUU
No ORF made
No ORF translated
Shine-Dalgarno-Sequestor
SD
SD
Anti-Sequestor
GENE ON GENE OFF
GENE ON GENE OFF
SDSDΔT No ORF
translatedOpen Thermometer
Closed Thermometer
Figure 1. Schematic model of gene regulation by riboswitches and RNA thermometers. See text for further information. (A) Transcription termination mechanism used by some riboswitches to
regulate gene expression. If the ligand (red circle) is not present during the transcription of the 5‘-UTR (GENE ON), an anti-terminator hairpin forms which does not interfere with expression of the open reading
frame (ORF). When present at elevated concentrations (GENE OFF), the ligand binds to the riboswitch which results in formation of a terminator hairpin. (B) A translation-inhibition mechanism of riboswitch gene
regulation. If the ligand is not present (GENE ON), an anti-sequestor stemloop forms which does not interfere with translation of the ORF. At elevated concentrations, the ligand binds to the riboswitch and allows
formation of a stemloop structure that sequesters the SD sequence and prevents the ribosome from binding to the mRNA. (C) Mode of action of RNA thermometers that control translation during a temperature shift. At
elevated temperature (GENE ON), the structure around the translation initiation region melts open and allows the ribosome to translate the ORF. At low temperature (GENE OFF), the SD site is base paired and
prevents the ribosome from translating the ORF. RNAP, RNA polymerase; SD, Shine Dalgarno; Δ T, temperature shift (Blount and Breaker 2006, revised)
A. Introduction
3
inner membrane protein synthesized under extreme alcaline stress (22). It is known that sequence-
specific pausing and the elongation speed of RNA polymerase during de novo synthesis influences
the dynamics of RNA folding (23). In vitro transcription assays demonstrated that the RNA
polymerase pauses at two distinct sites during alx transcript elongation under alcaline conditions
(21). The prolonged transcription pausing allowed subsequent formation of translatable messenger
intermediates. This was not the case under neutral pH conditions, when the riboswitch folded into
a translationally inactive conformation.
Already 20 years earlier, Shoshy Altuvia showed that translation efficiency of an RNA
segment can be dependent on temperature (24). Since then, it has become evident that both, non-
pathogenic and pathogenic microorganisms are able to regulate gene expression via RNA
thermosensors, or „RNA thermometers“ (25). These leader sequences fold in a manner that is
sensitive towards temperature alterations. With increased temperature, certain occluded regions,
i.e. the SD sequences, are liberated and translation can initiate (Fig. 1c). Usually, RNA
thermometers are found upstream of genes that are more or less directly related to temperature,
encoding either heat shock proteins or virulence regulators (26-29). However, the first described
temperature-responsive RNA element regulates viral lysis-lysogeny decision. The cIII RNA
segment in the bacteriophage λ was present in two mutually RNA secondary structures, A
(TRANSLATION OFF) and B (TRANSLATION ON), at equilibrium (24; Fig. 2a). At temperatures
below 37°C, the equilibrium shifted to structure B, causing accessibility of the SD site due to an
interaction between the anti-SD site and the anti-anti-SD site. The unpaired SD site facilitated
binding of the 30S subunit and translation of the cIII mRNA which in turn positively regulated the
lysogenic pathway. With an increase in temperature to 45°C, the structure was rearranged and the
equilibrium shifted to structure A. The SD site in structure A was sequestered by base pairing with
the anti-SD site, which prevented synthesis of the cIII protein and drove the bacteriophage λ into
the lytic cycle. It is noteworthy, that the cIII thermosensor deviates from other RNA thermometers in
that translation of cIII transcript is reduced with increasing temperature.
3. The E. coli rpoH thermometer
A sudden increase in temperature results in misfolding and accumulation of denatured proteins,
culminating in a global cellular response and stimulation of the multilayered heat shock regulon
A. Introduction
4
(30). This process was shown to be controlled mainly on transcriptional level by repressor proteins
and alternative sigma factors (31). The concerted expression of genes that maintain E. coli growth
under high temperature stress requires the expression of sigma factor σ32, encoded by the rpoH
gene. Interestingly, expression of σ32 itself is, to a great extent, governed on posttranscriptional and
translational level (32). Through various experiments, it was shown that two separated segments of
the rpoH transcript coding region (nucleotides 1 to 27 and 112 to 208) fold into a complex structure
that prevented translation initiation at low temperatures (32, 33; Fig. 2b). The rpoH thermometer is
the most complex heat shock sensor described to date, not only due to its structure but also
because of a sophisticated melting mechanism. An increase in temperature results in weakening of
the structure and concomitant liberation of a sequence downstream of the AUG start codon that
showed complementarity to a part of the 16S rRNA . This is the primary ribosome docking site
which facilitates subsequent binding of the 30S subunit to the SD sequence and translation
initiation. In fact, there is evidence that transient binding to non-SD sites („ribosome standby sites“)
is crucial to recruit ribosomes from the cytoplasm (34). Synthesis of the RpoH sigma factor then
induces transcription of a large heat shock regulon.
4. ROSE - Repression Of heat Shock gene Expression
The by far most common class of RNA thermometers are the ROSE (Repression Of heat Shock
gene Expression) elements. Shortly after its discovery in rhizobia (26, 35) it has been identified as
a conserved regulatory element in α and γ-proteobacteria (36). With a length from 60 to more than
100 nucleotides, the ROSE thermometers are comprised of two, three or four different hairpins.
While the 5‘ hairpin(s) remain(s) stable under heat shock conditions (37), the SD-sequestering 3‘
proximal hairpin is only stable at low temperatures (38). Temperature induced local melting in the 3‘
proximal hairpin exposes the SD site, facilitating ribosome binding (Fig. 2c). The other hairpins
probably ensure proper folding during de novo synthesis or alternative, to fold into a tertiary
structure supporting translation initiation at elevated temperatures (26, 38). Common to all ROSE
thermometers is the presence of a nucleotide stretch in close vicinity to the SD sequence. All
ROSE elements share a U-U/C-G-C-U motif. The centrally preserved G was first believed to be
unable to base pair and therefore cause a bulge opposite the SD sequence (38). However, recent
A. Introduction
5
NMR studies have shown that the G is not exposed as predicted but paired in a syn-anti
conformation with a second G residue of the SD region (39). A 29 nucleotide fragment carrying the
conserved ROSE sequences was used for three-dimensional structure determination and melting
studies. NMR studies showed that local melting of the RNA structure initiates from an internal loop
harbouring a U-U pair and proceeds into the SD sequence. Deletion of the G residue in the ROSE1
thermometer of Bradyrhizobium japonicum or the ibpA thermometer of E. coli completely abolished
Figure 2. Schematic model of temperature-dependent sequestration of the ribosome binding site in different RNA thermometers. See text for further information. (A) Structure A: Docking of the ribosome (grey circles) to the SD sequence (red box) of the bacteriophage λ cIII messenger is hindered by a secondary structure at high temperature
(45°C). Structure B: At lower temperature (<37°C), a conformational change in the RNA structure facilitates binding of the ribosome to the SD sequence and translation initiation at the AUG start codon (black box). (B) Translation initiation of the E. coli rpoH mRNA depends on an interaction between the 16S rRNA and an element (grey box) in the rpoH coding region. At low temperatures (<30°C) the downstream element is not
accessible, preventing binding of the ribosome. With an increase in temperature (42°C) the RNA conformation is altered, facilitating ribosome binding to the downstream element, interaction of the ribosome
with the SD site and concomitant translation initiation. (C) The translation of many small heat shock gene mRNAs is prohibited at low temperatures (30°C) due to a stable interaction between the SD and the anti-SD
sequence, preventing ribosome binding. The SD sequence is released at elevated temperature (42°C), allowing binding of the ribosome and translation initiation. ΔT, temperature shift
5‘
5‘
5‘ 5‘
5‘
A λ cIII
B rpoH
C ROSE
ΔT
ΔT
ΔT
5‘
G G
A B
A. Introduction
6
translation induction at higher temperatures, suggesting that the non-canonically paired G residue
is crucial for melting of the SD sequence (26, 36). As it was the case for the rpoH thermometer, the
E. coli ibpA ROSE element is embedded within a multifactorial regulatory network, including
RNaseE-mediated processing events and translational regulation of the gene products by the Lon
protease (29, 40).
5. Cold shock thermosensors
Deletion analysis in the 5‘-UTR of cspA, the paradigm E. coli cold shock gene, indicated that cold
induction is mainly regulated on the posttranscriptional level by cis-acting elements of its transcript
(41). Recently, it was shown that at low temperatures, the „cold shock“ structure was more
efficiently translated and somewhat less susceptible to degradation than the 37°C structure (42). In
contrast to other thermosensors, the underlying conformational rearrangements did not result from
melting of hairpin structures. The low temperature conformation of cspA mRNA imposes structural
constraints that expose the SD sequence and place the start codon in an unstable helix.
Conversely, at 37°C these translation initiation elements are buried within a double-stranded
structure, a condition expected to limit translational efficiency. It was demonstrated by various
structure probing experiments that temperature alterations were sensed by the whole cspA mRNA
and that the cold shock response is dependent on the mRNA folding process.
6. RNA thermometers in bacterial pathogens
An increase in temperature to 37°C is a highly significant signal for a pathogenic microbe that it
has successfully invaded a warm-blooded mammalian host (43). To avoid the innate immune
response and to safe energy, pathogens express virulence genes shortly before starting host
invasion (44). Expression of the Yersinia pestis adhesion protein YadA requires the virulence factor
LcrF, which is present only at 37°C but not at 26°C (45). Already in 1993, Hoe and Goguen
predicted that the 5‘-UTR of lcrF might confer translational control to the virulence regulator gene
(46). Unfortunately, no further experiments were performed to validate this model. Yersinia
pseudotuberculosis contains a similar potential RNA thermometer upstream of its lcrF gene. In a
side project of this PhD thesis in collaboration with Katja Böhme and Petra Dersch (HZI,
A. Introduction
7
Braunschweig), we provided evidence that it controls ribosome access and permits virulence gene
induction at 37°C (data not shown).
A typical RNA thermometer controls expression of the Salmonella enterica serovar
Typhimurium gene agsA (27; Fig.3a). The agsA gene does not encode a virulence factor but a
molecular chaperone. It is known that the expression, proper folding and secretion of virulence
factors require the presence of certain chaperones that are upregulated due to severe stresses
during host invasion (47). RNA secondary structure prediction programs and RNA-probing
experiments revealed that the agsA 5‘-UTR formed two distinct hairpins, with the second hairpin
having the SD site sequestered by binding to a consecutive stretch of four uridines (27).
Toeprinting assays confirmed that formation of the ternary translation initiation complex (mRNA,
30S ribosomal subunit and tRNAfmet) occurs only at 45°C but not at 30°C. Certain hairpin-
A agsA
B prfA
5‘ΔT
ΔT
5‘4U 4U
5‘ 5‘
Figure 3. Schematic model of RNA thermometers in pathogenic bacteria. See text for further information. (A) Translation of the S. typhimurium agsA mRNA at low temperatures
(30°C) is inhibited by a SD site (red box) interaction with four uridines (4U), that hampers the binding of the ribosome (grey circles). At higher temperatures (42°C), the SD site is released, facilitating binding of the
ribosome and translation initiation. (B) Translation initiation of the L. monocyogenes transcriptional activator prfA at low temperatures (<30°C) is hindered by a secondary structure that masks the SD site. At elevated
temperatures (37°C), the secondary structure is partially disrupted, enabling docking of the ribosome and translation initiation. While translation of agsA contributes to proper functionality of expressed virulence
factors, translation of the prfA mRNA directly activates virulence genes. ΔT, temperature shift
A. Introduction
8
stabilizing nucleotide substitutions located at the base of the anti-SD site were shown to close the
hairpin, thereby completely abolishing the heat induction of the agsA gene. Conversely,
destabilizing point mutations in the four uridine stretch prevented repression at 30°C. As the four
uridine anti-SD residues are also present in Y. pestis lcrF, Staphylococcus aureus groES and
Brucella melitensis dnaJ, there is strong evidence that „FourU“ elements are widely used for
control of bacterial heat shock and virulence genes (25). Due to its compact size of 58 nucleotides,
the agsA 5‘-UTR is perfect for NMR studies of the entire thermometer. Recent NMR data provided
insights into melting of the agsA FourU thermometer at nucleotide resolution (48). Intriguingly, the
agsA 4U motif is not only relevant for imperfect pairing of the SD sequence, but also comprises an
experimentally verified Mg2+-binding site (Fig.4; Rinnenthal et al. 2010 in preparation).
In most cases, virulence genes are organized in a cascade-like fashion with a
transcriptional master activator on top of it (49). Upstream of the Listeria monocytogenes PrfA
virulence regulator encoding mRNA lies a 127 nucleotide-long 5‘-leader, which forms a complex
secondary structure (28). Despite the SD sequence and AUG start triplett being in exposed loops,
Figure 4. The Salmonella agsA FourU motif comprises a Mg2+ binding site.NMR spectroscopic analysis revealed that agsA thermometer-melting at elevated temperatures (42°C) is
based on Mg2+-binding to the U-G ensembles of the SD site (encircled). Presence of the cation resulted in enhanced stability of the structure. (Rinnenthal et al. in preparation, revised)
A. Introduction
9
the overall structure is sufficient to inhibit formation of the translation initiation complex at low
temperatures. The structure is destabilized at human body temperature (37°C) allowing translation
of the prfA messenger (Fig.3b). The presence of PrfA protein activates the expression of several
virulence genes, encoding not only adhesins, but also phagosome-escape factors and several
immune-modulating factors which repress the host innate response (50, 51). Base substitution
mutations that stabilized or destabilized the prfA-UTR structure, displayed a repressed expression
at 37°C and a derepressed expression at low temperatures, repsectively, both in E. coli and L.
monocytogenes (28). The complexity of prfA-regulation was further increased by recent findings of
the Johansson and Cossart labs, that a S-adenosylmethionine (SAM) riboswitch can function both
as a classical cis-acting riboswitch as well as a trans-acting sRNA by targeting the prfA
Figure 5. The truncated riboswitch element, SreA, interacts with the prfA-UTR at 37°C.See text for further information. The SAM riboswitch element A (SreA) can function in trans, by binding to the
prfA thermometer. The SreA:prfA-UTR interaction leads to a diminished expression of PrfA. The exact mechanism by which this interaction hamperes ribsome-binding remains to be elucidated. (Loh et al. 2009,
revised)
SreA:prfA-UTR
SreA prfA
5‘ 5‘
5‘
5‘
37°C
?
A. Introduction
10
thermometer (52). Transcription of the riboswitch is terminated by elevated levels of SAM. The
truncated element, SreA (SAM riboswitch element A, 227 nucleotides), directly interacts with the
distal part (80 bases upstream of the SD site) of the prfA thermosensor, thereby blocking
translation of prfA mRNA (Fig. 5). This interaction region is unusually distant from the SD site and
the precise mechanism of SreA-mediated translational inhibition remains unclear. The interaction
only occurs at elevated temperatures (37°C), when the prfA thermometer is in a more open
conformation. SreA expression itself is controlled by PrfA, forming a regulatory feedback loop.
Deletion of SreA resulted in upregulation of PrfA at 37°C, suggesting that SreA antagonizes Listeria
virulence. This „double“ regulation is an intelligent way for a pathogen to ensure translation of an
important protein under two different conditions (47). In theory, all transcriptionally controlled
riboswitch elements may function as sRNAs and regulate certain target mRNAs, which
dramatically increases the number of putative trans-acting sRNAs.
A. Introduction
11
B. Objectives
There is evidence that both pathogenic and non-pathogenic bacteria use RNA thermometers to
govern heat shock and virulence gene expression. Although the regulatory principle of translational
control by opening and closing of the thermometer appears very simple, we are still far from
understanding the molecular details of this reversible melting process. Internal loops and bulges
and non-canonical base pairs contribute to melting in the physiological range. Most RNA
thermometers are able to operate without additional cellular factors. However, in some cases
trans-active factors are involved in RNA thermometer function.
Intensive bioinformatic studies on RNA thermometers suggest that these regulatory
elements are widespread among prokaryotes. Hence, it is important to elucidate the true
physiological relevance of selected RNA thermometer candidates within complex stress and
virulence control networks. In the course of this thesis, we plan to address the following
fundamental questions:
- Is it possible to design a synthetic RNA thermosensor by the help of computer-based rational
design and in vivo selection?
- What is the physiological relevance of a simple RNA thermometer upstream of the
Synechocystis sp. PCC6803 small heat shock protein gene hsp17?
- How does a putative FourU thermometer control virulence gene expression in the human
pathogen Vibrio cholerae?
To address these issues, we will use both available molecular biological techniques and establish
novel methods to characterize the candidate RNA thermometers, e.g. chemical structure probing
(lead) along a temperature gradient, a gfp-reporter system to examine thermometer function, and
chromosomal insertion of repressed and derepressed thermometer variants to detect phenotypic
variations. Biophysical analyses and in vivo animal models will be conducted in close collaboration
with other laboratories.
B. Objectives
12
C. Generation of synthetic RNA-based thermosensors
Torsten Waldminghaus, Jens Kortmann, Stefan Gesing
and Franz Narberhaus
C. Generation of synthetic RNA-based thermosensors
13
Biol. Chem., Vol. 389, pp. 1319–1326, October 2008 • Copyright � by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.150
2008/166
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Generation of synthetic RNA-based thermosensors
Torsten Waldminghaus, Jens Kortmann,Stefan Gesing and Franz Narberhaus*
Lehrstuhl fur Biologie der Mikroorganismen, Ruhr-Universitat Bochum, D-44780 Bochum, Germany
* Corresponding authore-mail: franz.narberhaus@rub.de
Abstract
Structured RNAs with fundamental sensory and regula-tory potential have been discovered in all kingdoms oflife. Bacterial RNA thermometers are located in the 59-untranslated region of certain heat shock and virulencegenes. They regulate translation by masking the Shine-Dalgarno sequence in a temperature-dependent manner.To engineer RNA-based thermosensors, we used a com-bination of computer-based rational design and in vivoscreening. After only two rounds of selection, severalRNA thermometers that are at least as efficient as naturalthermometers were obtained. Structure probing experi-ments revealed temperature-dependent conformationalchanges in these translational control elements. Ourstudy demonstrates that temperature-controlled RNAelements can be designed by a simple combined com-putational and experimental approach.
Keywords: regulatory RNA; riboswitch; RNAthermometer; synthetic biology; translational control.
Introduction
Examples of naturally occurring RNA sensors are accu-mulating in the last years (Winkler and Breaker, 2005;Narberhaus et al., 2006). Usually, they are located in the59-untranslated region (59-UTR) of mRNAs, fold into com-plex structures and control the expression of down-stream genes by signal-induced conformational changes.While RNA thermometers sense temperature as a phys-ical stimulus, riboswitches recognize chemical signals.Target molecules are bound by riboswitches with highspecificity and affinity. Gene expression is regulatedeither at the level of translation initiation, transcription ter-mination or RNA processing (Mandal and Breaker, 2004;Nudler and Mironov, 2004).
RNA sensors are comparatively simple regulatorydevices, as they do not require accessory proteins. It hasbeen speculated that they represent an ancient mode ofgene regulation (Vitreschak et al., 2004). Great effort hasbeen made to engineer different types of riboregulators,which may act as antisense RNAs, ribozymes or smallmolecule-binding aptamers (Isaacs et al., 2006; Gallivan,2007; Suess and Weigand, 2008). Incorporation of a the-ophylline aptamer into a designed helix in the 59-UTR of
a reporter gene by Suess and colleagues produced asynthetic riboswitch (Suess et al., 2004). Addition of the-ophylline caused a dose-dependent increase in geneexpression when this riboswitch was assayed in theGram-positive bacterium Bacillus subtilis. Theophyllinealso serves as inductor of a synthetic riboswitch thatactivates translation in Escherichia coli (Desai and Galli-van, 2004).
Construction of riboswitches includes several steps.The first step often is an in vitro selection procedure tosearch for binding of a desired ligand. In a second step,the regulatory potential of ligand binding is then analyzedin an in vitro activity assay or an in vivo system. In mostcases, time-consuming optimization steps are needed toyield the desired regulatory effect. The function of RNA-based regulators is to a great extent dependent on theirstructure. Computer-based prediction of RNA structuresfrom the primary sequence usually is quite reliable. Thismotivated studies using computer predictions to engi-neer riboswitches. Penchovsky and Breaker used aseries of programs to design RNA switches with differentBoolean logical functions (Penchovsky and Breaker,2005). Their system is based on allosteric ribozymeswhose activity is triggered by oligonucleotide binding.The binding region is located in a loop whose ligand-dependent conformation modulates the catalytic core ofa minimal hammerhead ribozyme.
Recently, RNA thermometers have been recognized asan attractive subject for engineering (Lee and Kotov,2007; Wieland and Hartig, 2007). Naturally occurringRNA thermometers undergo temperature-induced struc-tural changes (Narberhaus et al., 2006). All presentlyknown RNA thermometers control translation initiation. Inmost cases, entry to the ribosome binding site is blockedby complementary base pairs at low temperatures. Atincreasing temperatures, melting of the structure permitsribosome access. This simple regulatory principle can berealized by quite different RNA structures. Only a few dis-tinct families of RNA thermometers have been discov-ered so far. ROSE (rIepression oI f heat sIhock geneeIxpression)-like thermometers consist of several stem-loop structures and control expression of small heatshock genes in many a- and g-proteobacteria (Nocker etal., 2001; Waldminghaus et al., 2005). Thermal control isachieved by temperature-labile, non-canonical base-pairs in the SD region (Chowdhury et al., 2006). ThefourU thermometer has a simpler architecture (Wald-minghaus et al., 2007b). It regulates expression of theSalmonella heat shock gene agsA and is composed ofonly two hairpins spanning 57 nucleotides. While the 59-proximal hairpin remains stable up to 508C, the secondhairpin melts with increasing temperature in the physio-logical temperature range. The temperature responsiveelement harbors the SD sequence base-paired with astretch of fourU residues.
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Figure 1 Bioinformatic prediction of temperature-controlled riboswitches.(A) Schematic representation of the two alternative structures that the approach was based on. The dot-bracket annotations belowthe corresponding structures were used as input for the switch program. SD sequence (GGAGG) and the translation start sites (AUG)are framed or underlined, respectively. (B) Flow scheme of the individual steps used for in silico prediction. Computer programs areprinted in bold font.
As the underlying mechanism of temperature sensingis a gradual melting of a weak stem-loop structure ratherthan a switch between two mutually exclusive confor-mations, RNA thermometers are often considered asmolecular dimmers rather than ON/OFF switches. AnRNA element regulating phage l development is the onlynotable exception (Altuvia et al., 1989, 1991). The 59-UTRof the gene coding for cIII appears to be in equilibriumbetween two alternative conformations. Variations intemperature or Mg2q concentration shift the equilibriumbetween both structures and thereby alter accessibilityof the SD region.
To generate a temperature-responsive regulatory RNAelement de novo, we set out to combine simple archi-tectural characteristics of the fourU thermometer with aswitch-like mechanism as described for the cIII ther-mometer. By computational design and in vivo screening,we were able to construct an RNA element capable oftemperature-dependent induction of gene expression.The structural basis underlying this regulation was ana-lyzed on the molecular level and revealed insights intothermosensing by a synthetic RNA thermometer.
Results
Architecture and design of RNA-basedthermoswitches
The common principle of RNA-based thermosensors isthe temperature-controlled access of the ribosome to theSD sequence (Narberhaus et al., 2006). On the basis ofthis principle, we simulated two temperature-dependentalternative structures (Figure 1A). Both structures containthe sequence of an optimal SD sequence (Curry andTomich, 1988) and an AUG start codon in the appropriate
distance of 9 nucleotides. In the assumed OFF structure,both of these sequences are involved in base-pairing inan extended hairpin (Figure 1A). The predicted ON struc-ture is composed of two smaller hairpins. The SDsequence is positioned in an exposed loop and the AUGstart codon is single-stranded. To arrive at sequencespotentially capable of folding into these two structures inresponse to temperature, we made use of softwaredeveloped by Flamm and colleagues. The so-calledswitch program computes bistable RNA molecules witha predicted ability to switch between two given struc-tures (Flamm et al., 2001).
Both alternative structures were converted into thedot-bracket annotation (Figure 1A) before being fed intothe switch program. Temperatures of 308C and 428Cwere assigned as temperatures to the OFF and ONstates, respectively. The expected output was a list ofsequences that predominantly fold into the correspond-ing structures at the indicated temperature. As outlinedin Figure 1B, candidate sequences retrieved by theswitch program were processed through several in silicoselection steps by making use of programs provided bythe Vienna RNA folding package (Hofacker, 2003).
The switch program returned numerous sequences asbasis for further analysis. A selection of candidates outof approximately 300 predicted sequences is shown inFigure 2A. Sequence 1 (Seq1) passed all of the followingselection criteria (Figure 1B). First, the score was below1.0 (Figure 2A). The score is an estimation of the switchpotential. It summarizes multiple parameters. Lower val-ues indicate better switch potential as sequences withhigher values (Christoph Flamm, personal communica-tion). Second, the calculated melting curve for thesequence shows a sharp peak in the temperature rangebetween 308C and 428C (Figure 2A). Third, the RNAfold
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Figure 2 Output of the in silico prediction.(A) Assorted sequences derived from the switch predictions are based on the secondary structures shown in Figure 1A. SD sequencesand translation start sites are underlined. Sequence 1 (Seq1) and sequence 2 (Seq2) were selected for further analysis. The meltingcurve of Seq1, calculated with the RNAheat program of the Vienna RNA Package, is depicted to the right. According to this calculation,the OFF and ON states are separated by a narrow energy-maximum and both conformations are predicted to switch at around 358C.(B) Energy dot plot of Seq1 calculated with RNAfold. The black squares describe the equilibrium base pairing probabilities. Thesequence has two dominating conformations with energies of -11.7 (ON, left) and -12.2 kcal/mol (OFF, right). The SD sequence inthe dot plot is shaded in gray.
Figure 3 Expression of translational bgaB fusions to predictedRNA thermometers.(A) Schematic representation of the reporter gene fusion on plas-mid pBAD-bgaB and (B) expression analysis of bgaB fusions.The Salmonella fourU-bgaB fusion served as positive control.Cells were grown in LB medium to exponential phase at 308C.Immediately after addition of 0.01% (w/v) arabinose, the cultureswere heat-shocked to 428C for 30 min. The relative b-galacto-sidase activity is shown as average of three parallel measure-ments with the indicated standard deviations.
program predicted a similar probability of both alternativestructures. This is visualized by the comparable squaresizes in the energy dot plot (Figure 2B). Finally, RNA sec-ondary structure predictions at different temperaturessupported a conformational switch between 378C and428C (data not shown). Most predicted sequences failedto pass the first selection criterion and had a score above1.0. Apart from Seq1, only Seq2 conformed to all criteria.The ability of both sequences to function as translationalregulatory control elements was assessed in E. coli.
In vivo characterization of thermoswitch candidates
Translational reporter gene fusions to Seq1 and Seq 2were placed downstream of the arabinose-induciblepBAD promoter (Guzman et al., 1995), which allows fortemperature-independent control of transcription (Figure3A). Upon annealing of appropriate oligonucleotides(Table 1), the AUG start codons of Seq1 and Seq2 wereligated to the bgaB sequence. bgaB codes for a heatstable b-galactosidase (Hirata et al., 1984). As positivecontrol, we used the recently described Salmonella fourUthermometer (Waldminghaus et al., 2007b). In the pres-ence of 0.01% (w/v) of the inductor L-arabinose, theexpression in E. coli of both putative thermoswitchfusions was very low. This basal expression is clearlyabove background, which is zero since E. coli has nochromosomally encoded thermostable b-galactosidase.Repression was not relieved when cells were grown at428C (Figure 3B). In contrast, the positive control (fourU)
showed approximately three-fold induction at the elevat-ed temperature.
Thermosensor engineering
Since Seq1 was incapable of conferring temperaturecontrol to a reporter gene fusion, we established an in
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Table 1 Strains, plasmids and oligonucleotides used in this study.
Strain, plasmid or Relevant characteristic(s) or sequencea Source oroligonucleotide reference
StrainsEscherichia coli DH5a F-F80dlacZDM15D(lacZYAargF) U169deoRrecA1endA1hsdR17 (Hanahan, 1983)
(rK-mK
q)sup44thi-1 gyrA69
PlasmidspBAD-bgaB Translational bgaB fusion vector, bgaB: heat-stable b-galactosidase, AmpR (Waldminghaus et al.,
2007a)pBO681 Seq1 fusion in pBAD-bgaB This studypBO684b Seq1-C37U fusion in pBAD-bgaB This studypBO685 Seq1-C10U fusion in pBAD-bgaB This studypBO686 Seq1-C26G fusion in pBAD-bgaB This studypBO690 Seq1-C38U fusion in pBAD-bgaB This studypBO692 Seq1-C26U-UG39/40AA fusion in pBAD-bgaB This studypBO693 Seq1-C10U-G36A fusion in pBAD-bgaB This studypBO694 Seq1-C26U-U39A fusion in pBAD-bgaB This studypBO695 Seq1-C10U-C26U fusion in pBAD-bgaB This studypBO696 Seq1-G24A-G36U fusion in pBAD-bgaB This studypBO699 Seq2 fusion in pBAD-bgaB This studypBO472 FourU fusion in pBAD-bgaB (Waldminghaus et al.,
2007b)
OligonucleotidesRBS1_fw TTTCTCGTGCTTAACGATTTCTAGGCGTGGAGGTTGCCTGGTATGGICTAGC
(construction of pBO681)RBS1_rv CATACCAGGCAACCTCCACGCCTAGAAATCGTTAAGCACGAGAAAGIAATTC
(construction of pBO681)RBS3_fw TTTCGCATGTTGAGTGCTTTTCCCCTGGGGAGGGCAGGGGATATGGICTAGC
(construction of pBO699)RBS3_rv CATATCCCCTGCCCTCCCCAGGGGAAAAGCACTCAACATGCGAAAGIAATTC
(construction of pBO699)mutbgaBfw GTCTATAATCACGG (error-prone PCR)mutbgaBrv TCTTGCTCCAACTG (error-prone PCR)Fw-bgab-probe AGAGCAATGGCCAGAGGAAA (generation of Northern blot probe)Rv-bgab-probe TAATACGACTCACTATAGATCGGCAAAGAATCTGGAT
(generation of Northern probe)aIntroduced restriction sites are underlined.bThe RNA sequence (U instead of T) is given.
Table 2 Single and multiple nucleotide exchanges in Seq1obtained by error-prone PCR mutagenesis.
Mutation in Seq1 PCR conditions
C10Ua Seq1 as template; selected twice;(a) four-fold dGTP and(b) four-fold dATP (0.8 mM)
C37U Seq1 as template; selected twice;(a) four-fold dGTP and(b) four-fold dCTP (0.8 mM)
C26G Seq1 as template; four-fold dTTP(0.8 mM)
C38U Seq1 as template; eight-fold dTTP(1.6 mM)
C10U-C26U C10U as template; standard dNTPs(0.2 mM), MnCl2
C10U-G36A (HIM) C10U as template; eight-fold dATP(1.6 mM)
C26U-U39A Seq1 as template; standard dNTPs(0.2 mM), addition of 0.2 mM MnCl2
C26U-UG39/40AA See aboveG24A-G36U See aboveaThe RNA sequence (U instead of T) is given.
vivo screening system to convert it into a functional ther-mosensor. For this, we amplified the Seq1 fragment byerror-prone PCR. Different conditions, in which each oneof the four nucleotides dGTP, dATP, dCTP and dTTP wereused in four- or eight-fold excess, were used (see exper-imental procedures). The resulting PCR fragments werecloned into the pBAD-bgaB vector and transformed intoE. coli. Transformants were plated on Luria-Bertani agarplates containing L-arabinose as inductor and X-gal tomonitor b-galactosidase activity. Following overnightincubation at 308C, plates were transferred to 428C andthe color of the colonies was visually inspected for sev-eral hours. Colonies that turned blue were selected, andthe corresponding plasmids were isolated andsequenced. Four different single point mutations wereobtained (Table 2). Strikingly, the C10U mutation andC37U mutation were both selected twice from differentmutagenic conditions. This strongly suggests that thenumber of possible point mutations facilitating temper-ature-dependent regulation is limited.
Despite the clearly visible phenotype on plates, a qual-itative b-galactosidase assay of the four point-mutatedSeq1 variants showed only a minor increase of expres-sion at 428C as compared to 308C (Figure 4A). The factthat we were able to select for such subtle changes intemperature-dependent expression demonstrates the
high sensitivity of the X-gal based selection procedureand its suitability for an in vivo screening approach.
To further optimize thermal induction, we carried out asecond round of error-prone mutagenesis. Conditions of
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Figure 4 Effect of selected single (A) and multiple (B) mutationsin Seq1 on temperature-dependent expression.The experimental procedures are as described in Figure 3.Arrows indicate the template sequence from which the muta-tions in (B) originate. (C) Schematic representation of single andmultiple mutations in the proposed ON structure. Point-mutatedpositions are encircled. SD sequence (GGAGG) and translationstart site (AUG) are framed.
Figure 5 Translational control by selected RNA elements oftemperature-dependent expression of bgaB fusions to Seq1, theC10U and HIM (C10U, G36A) RNAs.(A) Temperature-dependent expression was monitored in E. coliDH5a. Cells were grown in LB medium at 308C and heat-shocked to 428C for 30 min before b-galactosidase activity wasmeasured. The results are the average of three independentmeasurements with the indicated standard deviations. (B) In par-allel, total RNA was extracted from the same E. coli cultures.Equal amounts were separated on a 1.2% denaturing agarosegel, transferred to a positively charged nylon membrane andimmobilized for subsequent hybridization. Northern blot experi-ments were carried out using digoxigenin-labeled RNA probesto detect bgaB transcripts.
higher mutagenic strength were used, namely an eight-fold excess of one nucleotide and the addition of MnCl2(see the materials and methods section for details). Seq1and the mutated variant C10U served as PCR templates.Cloning and selection were performed as above. Fivenew variants, four with two-point mutations and one withthree mutations, were obtained (Table 2). b-Galactosi-dase expression of the new constructs was slightly high-er at 308C compared to Seq1 or C10U (Figure 4B). Ineach case, the performance as thermosensor wasimproved, since expression increased approximatelytwo-fold (C10U-C26U and G24A-G36U) or approximatelythree-fold (C10U-G36A, C26U-U39A and C26U-UG39/40AA) at 428C.
Except for C10U, all selected mutations are located inthe second stem region of the ON structure (Figure 4C).Furthermore, all these mutations would destabilize thestem structure by either introducing a mismatch (G24A,C26G, G36U, G36A and U39A) or by changing a stable
GC base pair to a weaker GU pair (C26U, C37U, C38U).Taken together, these findings point to a critical role ofhairpin stability in thermosensing.
Temperature-dependent regulation is due totranslational control
Our experimental setup was designed to select for ther-mosensors that act as translational control elements.However, in principle the selected mutations might alsoaffect the mRNA level by altering RNA stability. To rulethis out, we repeated the b-galactosidase assay for Seq1and the two variants C10U and C10U-G36A (henceforthcalled HIM for High Induction Mutant). In parallel, we tooksamples for RNA isolation and subsequent Northern blotanalysis. RNA levels were monitored using a probedirected against the coding region of the bgaB reportergene (Figure 5). Ribosomal RNAs served as internal load-ing control. Although the recorded mRNA levels wereequivalent at 308C and 428C for all three constructs, b-galactosidase activity was slightly induced in the C10Umutant and highly induced in the HIM strain. Translationalrepression at 308C is illustrated by the finding that dif-ferent C10U and HIM mRNA levels resulted in essentiallythe same basal b-galactosidase activity (Figure 5). Tem-perature-induced expression despite constant mRNAlevels clearly supports a translational control mechanism.
Enzymatic probing experiments reveal structuralbasis for thermosensing
To address the structural requirements for thermosen-sing, we performed enzymatic probing experiments using
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Figure 6 Structure probing of synthetic Seq1 (A) and HIM (B)RNAs.RNase T1 (0.01 U) cleavage of 59-end-labeled RNAs was carriedout from 208C to 508C in intervals of 58C. RNA fragments wereseparated on 8% polyacrylamide gels. Lane C: incubation con-trols with water were taken at 208C, 358C and 508C. Lane L:alkaline ladder. Relevant G-residues are marked with arrow-heads to the right. The SD and stem sequences are labeled tothe left. Selected parts of SD and stem region are highlightedwith boxes. (C) Secondary structure model of Seq1 RNA con-sisting of hairpin I and II. RNase T1 cleavage sites are shownby arrows. Circled nucleotides mark exchanges that result in thethermoresponsive HIM RNA. SD sequence (GGAGG) and trans-lation start site (AUG) are framed.
RNase T1 which cuts selectively 39 of single-strandedguanine residues. We probed the structures of syntheticSeq1 and HIM RNAs at temperatures ranging from 208Cto 508C in intervals of 58C (Figure 6). The overall cleavagepattern of Seq1 at 208C is in good agreement with thecalculated ON structure (Figure 6C). G-residues in theterminal loop of hairpin II at positions 28–34, which con-tains the SD sequence, were cut by RNase T1. Con-versely, protection of regions 23–26 and 34–40 againstcleavage confirmed the predicted stem regions in hairpinII. Consistent with the low reporter gene activity, the RNAstructure was not temperature-responsive, as it remainedresistant against cleavage by RNases T1 at 408C andwas only moderately cleaved at 458C and 508C in thestem region at positions 24 and 25 (see box in Figure6A). The structure probing results clearly show that thecomputer-predicted switch between alternative struc-tures does not occur. Under our experimental conditions,the sequence does not fold into the OFF structure at all.
Also, the HIM structure does not seem to fold into thepredicted OFF structure. There are essentially no differ-ences between the structures of hairpin I in Seq1 andHIM (Figure 6). The predicted hairpin II of the HIM struc-ture, however, is less stable as in Seq1. The G36Aexchange results in an extended loop that is readilyaccessible to RNase T1, even at low temperature (Figure6B). The flanking regions are more resistant to thisenzyme at 208C, indicating a double-stranded RNA atlow temperature. Residual cleavage by T1 suggests thata minor population might be in an open conformation. Inaccordance with induced expression (Figures 4 and 5),the fraction of single-stranded residues substantiallyincreased with increasing temperature as illustrated bythe accumulation of T1-derived products at nucleotides24 and 25 (see box in Figure 6B and arrows in Figure6C).
Discussion
Heat-inducible systems have been shown to be suitablefor controlled expression of recombinant genes and var-ious industrial applications (Heitzer et al., 1992). Engineer-ing of RNA-based modules to achieve temperature-regulated gene expression is attractive for several reasons.First, cis-active RNA thermometers are independent ofadditional (protein) factors and therefore transferable tovarious biological systems. Second, there is no need forchemical inducers as in the case of riboswitches. Suchinducers often face technical drawbacks, such as poormembrane permeability or high costs in large scale appli-cations. Third, various computational tools have recentlybeen developed to support rational design of RNA ele-ments. Using a two-step approach composed of com-putational design and in vivo screening, we successfullyengineered RNA-based thermosensors.
Prediction of temperature-dependent RNA switches
RNA secondary structures are to a great extent com-posed of complementary base pairs. Algorithms to pre-dict the structure of a given RNA molecule based on themaximal number of base pairs possible have been devel-oped 30 years ago (Nussinov and Jacobson, 1980). Lat-er, experimental data were included to further optimizeprediction quality (Mathews et al., 1999). Matters arecomplicated by the fact that one RNA molecule can oftenfold into two or more different but energetically similarstructures. On the other hand, a given structure might beformed by RNAs with various sequences. Flamm andcolleagues developed a program to calculate sequencesable to fold into two given structures (Flamm et al., 2001).Since RNA folding is strongly temperature-dependent,this parameter was implemented in the program. Ourexperimental study, however, revealed that such theoret-ical assumptions might not necessarily be transferable toan in vitro or in vivo situation. The computer-derivedsequences Seq1 and Seq2 should have resulted in highreporter gene activities at elevated temperatures, whichwas not the case. Furthermore, determination of the RNAstructure of Seq1 in vitro (Figure 6) shows that it falls intothe predicted ON structure, regardless of the tempera-ture. Although the G-residues in the SD sequence in loop
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II were accessible to RNAse T1, the small loop was notsufficient to allow entry of the 30S ribosomal subunit,since translation was inefficient. Apparently, it remains achallenge to reliably predict RNA sequences that switchbetween two alternative structures.
A promising approach for RNA regulatorengineering
The power of in vivo screening systems has been widelyused to improve enzyme function and to develop geneticcontrol systems and RNA-based regulators (Buskirk etal., 2003; Haseltine and Arnold, 2007; Yuen and Liu,2007). Also, our study demonstrates that a functionalRNA regulator can be developed successfully by appli-cation of a suitable selection approach. Two findings areespecially remarkable. First, two out of four selected sin-gle-point mutations were selected independently, despitedifferent experimental conditions (Table 2). This mightreflect the limited possibilities to evolve a functional reg-ulator from the given input structures. Such a narrow win-dow for de novo design of a functional RNA thermometeris conceivable, since most RNA structures would beeither too tight or too loose to allow temperature controlin the physiological temperature range.
The second interesting finding is that the combinationof only two-point mutations turned the translation-incom-petent structure Seq1 into a synthetic thermosensor thatis as efficient as natural thermometers. Only minorchanges in the nucleotide composition can have drasticeffects on natural RNA thermometers. Deletion of a singleG-residue that introduces a labile structure in ROSE ele-ments results in a SD sequence that remains base-pairedeven at high temperatures (Chowdhury et al., 2003,2006). On the other hand, single-point mutations canlead to derepression at low temperatures as describednot only for the ROSE thermometer but also for the prfAthermometer of Listeria monocytogenes and the rpoHthermometer of E. coli (Morita et al., 1999; Johansson etal., 2002).
It remains a matter of speculation whether the shortpath to generate an RNA sensor in our study means thatthe starting structure was already close to being an RNAthermosensor or whether many different structures mightbe transformed into an RNA thermosensor by only a fewscreening steps. It is noteworthy that RNA thermometerswith different architectures and riboswitches, whichsense the same metabolite by different structures, havebeen described (Corbino et al., 2005; Narberhaus et al.,2006). Nevertheless, there might be a limitation in thenumber of possible RNA structures with the same func-tionality. In vivo screening approaches starting from ran-dom sequences could help to address how many optionsthere might be in nature.
Materials and methods
Plasmid construction and in vivo screening
To generate plasmids pBO681 and pBO699, oligonucleotidescorresponding to the predicted sequences were ordered (MWG-Biotech, Ebersberg, Germany) and cloned into the NheI andEcoRI restriction sites upstream of bgaB in pBAD-bgaB (Table
1). Plasmid pBO681 served as template for random mutagenesisby error-prone PCR using primers mutbgaBfw and mutbgaBrvto generate pBO684, 685, 686, 690, 692, 694 and 696 (Table 1).Plasmids pBO693 and 695 were generated with the same prim-ers and pBO685 as template. The error-prone PCR was per-formed by using Taq DNA polymerase in PCR buffer w20 mM TrisHCl (pH 8), 10 mM KCl, 6 mM (NH4)2SO4, 2 mM MgSO4=6H2O,0.1% Triton X-100x, 25 pmol of each primer, 3 mM MgCl2 and anucleotide concentration of 0.2 mM each in a total volume of50 ml. To favor misincorporation of nucleotides, the followingmodifications to the standard protocol were used. One of thefour nucleotides dGTP, dATP, dCTP and dTTP were used in four-(0.8 mM) or eight-fold (1.6 mM) excess. In some cases, 0.2 mM
MnCl2 were added in the presence of standard nucleotide con-centration. The PCR program was as follows: initial DNA dena-turation at 948C for 2 min was followed by 35 cycles at 948C for30 s, primer annealing at 408C for 30 s and elongation at 728Cfor 30 s.
PCR products were digested with EcoRI and NheI, cloned intothe corresponding sites upstream of bgaB in pBAD-bgaB andtransformed into E. coli DH5a. Cells were grown on LB agarplates containing X-gal (40 mg/ml) in the presence of 0.01%(w/v) L-arabinose overnight at 308C. Following a temperatureshift to 428C, candidates that turned blue earlier than otherswere collected and the assay was repeated before the b-galac-tosidase activity was measured at 308C and 428C. Plasmidsfrom clones with elevated bgaB expression at 428C were isolat-ed and sequenced.
b-Galactosidase assay
E. coli cells carrying bgaB fusions were grown to exponentialgrowth phase at 308C. Samples of 10 ml were transferred to428C for 30–60 min, before b-galactosidase activity was meas-ured as described previously (Miller, 1972), except that enzymeactivity was measured at 558C. The b-galactosidase activity inFigures 3B and 4A is denoted relative to the activity of Seq1-bgaB at 308C and in Figure 4B relative to the parental construct(Seq1-bgaB for the first three and C10U-bgaB for the last two)at 308C.
Isolation of RNA and Northern blot analysis
Total RNA was extracted from E. coli cells using the hot phenolmethod (Aiba et al., 1981). Equal amounts of total RNA samples(5 mg) were separated on a 1.2% formaldehyde-agarose gel,transferred to a positively charged nylon membrane (Hybond N1;Amersham Biosciences, Buckinghamshire, UK), and hybridizedat 688C using a DIG-HIGH prime labeling and detection kit asper the manufacturer’s protocol (Roche Applied Science, Mann-heim, Germany). The probe used was a 240-bp RNA fragmentcorresponding to the bgaB ORF. Detection was carried out byexposing the blot to a luminescence detector using chemilu-minescence substrate (CSP-D-Star, Roche Molecular Bioche-micals, Mannheim, Germany).
Structure probing experiments
RNA oligomers for structure probing experiments were pur-chased from Vbc biotech (Vbc-biotech GmbH, Vienna, Austria).Partial digestions of 59-end-labeled RNAs with ribonuclease T1were conducted as follows. 30 000 cpm of labeled RNA weremixed with 1 ml 5= TMN buffer (100 mM Tris acetate, pH 7.5,10 mM MgCl2, 500 mM NaCl) and 0.4 mg tRNA, and distilledwater was added to a volume of 4 ml. Samples were pre-incu-bated for 5 min at the indicated temperature, before 1 ml of T1RNase (0.01 U) was added. After 5 min of cleavage, 5 ml for-mamide loading dye was added and the samples were heatedat 958C for 5 min prior to separation on denaturing 8% poly-
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acrylamide gels. Alkaline ladders were generated as describedpreviously (Brantl and Wagner, 1994).
Acknowledgments
We thank Christoph Flamm (University of Vienna, Austria) forproviding us with the switch program and for help with com-puting. The work was funded by grants from the GermanResearch Foundation (NA 240 and SPP 1258) to F.N. and a fel-lowship from the Studienstiftung des Deutschen Volkes to J.K.
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Received April 30, 2008; accepted June 25, 2008
D. Translation on demand by a simple RNA-based thermosensor
Jens Kortmann, Simon Sczodrok, Jörg Rinnenthal, Harald Schwalbe
and Franz Narberhaus
submitted
D. Translation on demand by a simple RNA-based thermosensor
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1
Translation on demand by a simple
RNA-based thermosensor
Jens Kortmann, Simon Sczodrok, Jörg Rinnenthal†, Harald Schwalbe† and Franz
Narberhaus1
Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, 44780 Bochum,
Germany
†Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic
Resonance, Johann Wolfgang Goethe-University, 60438 Frankfurt/Main, Germany
Running title: Cyanobacterial RNA thermometer
Keywords: riboregulator; RNA thermometer; post-transcriptional control; heat shock;
photosynthesis; Hsp17
1Corresponding author: Franz Narberhaus, Lehrstuhl für Biologie der Mikroorganismen,
Ruhr-Universität Bochum, Universitätsstrasse 150, NDEF 06/783, 44780 Bochum, Germany.
Tel: +49 (0)234 322 3100; Fax: +49 (0)234 321 4620; E-mail: franz.narberhaus@rub.de
D. Translation on demand by a simple RNA-based thermosensor
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Structured RNA regions are important gene control elements in pro- and eukaryotes.
Here, we show that the mRNA of a cyanobacterial heat shock gene contains a built-in
thermosensor critical for photosynthetic activity under stress conditions. The
exceptionally short 5’-untranslated region is comprised of a single hairpin with an
internal asymmetric loop. It inhibits translation of the Synechocystis hsp17 transcript
at normal growth conditions, permits translation initiation under stress conditions and
shuts down Hsp17 production in the recovery phase. Point mutations that stabilized
or destabilized the RNA structure deregulated reporter gene expression in vivo and
ribosome binding in vitro. Introduction of such point mutations into the Synechocystis
genome produced severe phenotypic defects. Reversible formation of the open and
closed structure was beneficial for viability, integrity of the photosystem and oxygen
evolution. Continuous production of Hsp17 was detrimental when the stress declined,
indicating that shutting-off heat shock protein production is an important, previously
unrecognized function of RNA thermometers. We discovered a simple biosensor that
strictly adjusts the cellular level of a molecular chaperone to the physiological need.
D. Translation on demand by a simple RNA-based thermosensor
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Introduction
Cyanobacteria are ubiquitiously distributed on earth and - together with plants - provide the
foundation of aerobic life by the photosynthetic generation of oxygen. The integrity of the
photosynthesis machinery is challenged by highly fluctuating environmental conditions. In
particular, heat, high light intensities, reactive oxygen species, salt and metal stress are
known to cause defects of the thylakoid membrane-associated photosystems (1,2).
The small heat shock protein Hsp17 (also known as Hsp16.6 or HspA) is essential for
stress tolerance in the model cyanobacterium Synechocystis sp. PCC 6803 (3,4). Hsp17
belongs to the ubiquitous family of α-crystallin-type ATP-independent chaperones (5). Small
heat shock proteins (sHsps) capture unfolded proteins to prevent formation of irreversible
aggregates (6). Synechocystis Hsp17 not only possesses protein-protective activity but also
stabilizes the lipid phase of membranes, thus maintaining thylakoid membrane integrity
under stress conditions (7).
The exposure of Synechocystis to a sudden increase in temperature or light intensity
triggers expression of the heat shock regulon including hsp17 (3,8). Shifting Synechocystis
cells from 34°C to 44°C results in a more than 60-fold induction of hsp17 mRNA (9). Global
gene expression profiling revealed a 20-fold induction of the hsp17 transcript under light
stress (8). Transcription of heat shock genes, including hsp17, was shown to rely on the
alternative sigma factors SigB and SigE (10,11). Furthermore, hsp17 transcription is strongly
regulated by changes in the physical order of membranes (12). A combined transcriptomics
and proteomics approach suggested that regulation of heat shock gene expression in
Synechocystis is governed by transcriptional and yet unknown translational regulation
(9,11,13,14).
In recent years, the universal importance of regulatory RNAs as posttranscriptional
gene control elements has been recognized (15,16). In bacteria, small regulatory RNAs
(sRNAs) are very abundant regulators that often act through base-paring with target mRNAs,
thereby modulating translation efficiency and mRNA stability (17,18). Biocomputational
predictions and experimental strategies have revealed several hundred sRNAs in
D. Translation on demand by a simple RNA-based thermosensor
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Synechocystis; some acting as trans-encoded regulatory RNAs with short and discontinuous
complementary to their targets, others as cis-encoded perfectly complementary antisense
RNAs (19-21).
In contrast to numerous sRNAs mRNA-inherent riboregulators like riboswitches and
RNA thermometers have received little attention in cyanobacteria. Riboswitches are mRNA
leader sequences that fold into a complex structure whose conformation changes upon
ligand binding (22). RNA thermometers are translational control elements built-into the 5’-
untranslated region (5’-UTR) of bacterial heat shock or virulence genes (23). Typically, they
fold into a complex structure that traps the Shine-Dalgarno (SD) sequence at low
temperatures. An increase in temperature to 37°C (virulence genes) or higher (heat shock
genes) destabilizes the structure, liberates the SD sequence and permits formation of the
translation initiation complex. Functionality of RNA thermometers in the relevant temperature
range of mesophilic bacteria requires a structure that is stable enough to resist opening at
temperatures below 30°C but sufficiently unstable to melt as the temperature increases. This
can be achieved by a delicate balance of Watson-Crick base pairs, internal bulges and loops
and non-canonical base pairing (24-27).
An RNA thermometer-like structure is located in the 5’-UTR of the Synechocystis
hsp17 transcript. The hairpin engages the SD sequence and part of the AUG start codon in a
secondary structure, contains an internal loop and might thus act as RNA thermometer (Fig.
1A). With only 44 nucleotides in length, the hsp17 5’-UTR is the smallest natural
thermometer candidate discovered yet. In this work, we provide genetic and biochemical
proof that it acts as bona fide RNA thermometer that has important not previously described
physiological functions.
D. Translation on demand by a simple RNA-based thermosensor
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Materials and methods
Strains and growth conditions
E. coli cells (DH5α and DH5αZ1) were grown at 28 or 37°C in Luria-Bertani (LB) medium
supplemented with ampicillin (Ap, 150 µg/ml) or chloramphenicol (Cm, 50 µg/ml). For
induction of the pBAD promoter in strains carrying translational bgaB fusions, 0.01% (w/v) L-
arabinose was added. Expression of translational gfp fusions was induced via inactivation of
the Tet repressor with 50 ng/ml doxycycline.
Synechocystis cells were grown under low light conditions (30 µmol photons m-2s-1) at 28°C
on BG11/agar (28) plates or in BG11 liquid media as described (29). Liquid media was
supplemented with 5 mM glucose when appropriate.
For the measurement of chlorophyll concentrations, cells were sedimented by centrifugation
and extracted with 100% methanol. The concentration of chlorophyll was calculated from the
absorbance values of the extract at 666 and 750 nm (30).
Plasmid construction
The hsp17-bgaB fusion (pBO1293) was constructed by transfer of the hsp17 5’-UTR from
pBO1292 upon NheI/EcoRI digestion into the corresponding site in pBO415 (Table S1). To
obtain the translational gfp fusion (pBO1325), the hsp17 5’-UTR was transfered from
pBO1292 via NheI and an introduced PstI site into pXG-10 (31). Site-directed mutagenesis to
generate pBO1312, 1310, 1311, 1316, 1314, 1315, 1313, 1801 and 1802 was performed
according to the instruction manual of the QuikChange® mutagenesis kit (Stratagene, La
Jolla, USA). Plasmid pBO1292 served as a template for PCR with mutagenic primers (Table
S1). The inserts containing mutated hsp17 UTRs were isolated upon NheI/EcoRI or PstI/
NheI digestion and cloned into the corresponding site upstream the bgaB or gfp gene,
respectively. The entire hsp17 coding region including its 5’-UTR was amplified and cloned
into pUC18 (pBO1347) for subsequent site directed mutagenesis (Table S1). Using unique
restriction sites BamHI/CpoI, hsp17 thermometer variants rep, derep and WT were cloned
into pNaive.16 (29) resulting in pBO1834, 1806 and 1807. The correct nucleotide sequence
D. Translation on demand by a simple RNA-based thermosensor
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was confirmed by automated sequencing (eurofins, Martinsried, Germany). pNaive.16
(pAZ877) was provided by Prof. Dr. Elisabeth Vierling (University of Arizona).
RNA and protein detection
Preparation of total RNA from E. coli and Northern analysis followed previously published
protocols (32). Preparation of total RNA from Synechocystis was performed with a Qiagen
RNeasy® kit (Qiagen, Hilden, Germany) according to the instruction manual. RNAs were
detected with a DIG-labeled bgaB or hsp17 RNA probe, respectively. The DIG-HIGH prime
labeling kit (Roche Applied Science, Mannheim, Germany) was used for preparation of
probes (see Table S1 for details). For E. coli protein extraction, cell pellets were resuspended
in lysis buffer (10 mM sodiumphosphate, pH 7.0, 20% glycerol, 0.5 mM DTT) according to
their cell density (100 µl buffer per OD580 of 1.0). SDS sample buffer was added, cells were
boiled for 5 min and equal volumes were subjected to SDS-PAGE. Synechocystis whole cell
lysates were prepared according to Klinkert et al. (33). Soluble fractions corresponding to 20
µg of chlorophyll were separated on 12.5% polyacrylamide gels. SDS-PAGE and Western
blot was performed as described (34). GFP was detected on a Western blot using a
polyclonal α-GFP antibody (abcam209; Abcam, Cambridge, USA). Hsp17 antigen was
revealed by α-Hsp17 antibody, kindly provided by Prof. Dr. Elisabeth Vierling (University of
Arizona). Signals were detected with a ChemiImager™ Ready (Alpha Innotech, Biozym,
Wien, Austria).
In vitro transcription
RNAs were synthesized in vitro by runoff transcription with T7 RNA polymerase from
linearized plasmid templates. Plasmids pBO1301, 1304 and 1302 (linearized with MlsI) were
used to generate RNA for enzymatic structure probing and plasmids pBO1305, 1349 and
1348 (linearized with HpyCH4V) to generate RNA for toeprinting experiments (Table S1).
Structure probing experiments
RNA structure formation at either 28 and 42°C was compared. RNA was 5’-end labeled as
described (35). Partial digestion of 5’-end-labeled RNAs with ribonucleases T1 (0.004U and
D. Translation on demand by a simple RNA-based thermosensor
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0.01U), V (0.008U and 0.02U) was conducted according to Waldminghaus et al. (32). For
chemical probing, labelled RNA was mixed with 2 µl 5x lead buffer (250 mM tris acetate pH
7.5, 25 mM Mg acetate; 250 mM Na acetate) and 1 µ g tRNA in a total volume of 10 µl.
Lead(II) probing assay was conducted as described (36). RNA fragments were separated on
denaturing 8% polyacrylamide gels. Alkaline ladders were generated as described previously
(35).
Toeprinting analysis
Primer extension inhibition experiments were carried out using 30S ribosomal subunits,
target mRNA and tRNAfMet basically according to (37). The 5’-[32P]-labeled hsp17-specific
oligonucleotide hsp17therm-runoff-toeprint (Table S1) was used as a primer for cDNA
synthesis. An aliquot of 0.08 pmol mRNA (5’-UTR plus 63 nt of the hsp17 coding region)
annealed to the oligonucleotide was incubated for 10 min at 28 or 42°C, respectively,
together with 16 pmol of uncharged tRNAfMet (Sigma-Aldrich, St. Louis, Missouri). 6 pmol of
30S subunits or water (negative control) was added and incubation for another 10 min
followed before 2µl of MMLV-Mix (VD+Mg2+-buffer, BSA, dNTPs and MMLV reverse
transcriptase (USB, Cleveland, Ohio)) was added. cDNA synthesis was performed at 28°C.
Reactions were stopped after 10 min by adding formamide loading dye and aliquots were
separated on a denaturing 8% polyacrylamide gel.
CD spectroscopy
Circular dichroism (CD) unfolding and refolding curves were recorded with a JASCO
spectropolarimeter J-810. RNA concentration was adjusted to 25 µM. Buffer
conditions: 15 mM KxHy(PO4), 25 mM KCl, pH 6.5. CD unfolding curves were recorded with a
temperature slope of 1°C/min at a wavelength of 263 nm between 5°C-90°C. CD refolding
curves were recorded with a temperature slope of -1°C/min starting from 90°C to 5°C. The
CD melting curves were normalized to determine the fraction of the unfolded RNA α(T)
according to Equation (1).
D. Translation on demand by a simple RNA-based thermosensor
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(1)
In Equation (1) T is the temperature, θ(T) the ellipticity in [mdeg], θunfolded(T) the ellipticity
of the unfolded RNA, θfolded(T) the ellipticity of the folded RNA and α(T) the temperature
dependent fraction of unfolded RNA (RNAunfolded) with respect to the total amount of RNA
(RNAtotal). At the melting temperature Tm the fraction of unfolded RNA is α(Tm) = 0.5.
The normalized CD unfolding and refolding curves were fitted according to Hill’s equation
In Equation (2) Tm is the melting temperature of the RNA, a the amplitude, T the temperature
and b the Hill coefficient. The Hill coefficient is a measure for the cooperativity of the RNA
unfolding transition. High Hill coefficients indicate sharp unfolding transitions while low Hill
coefficients indicate a gradual transition from the folded to the unfolded conformation.
β-Galactosidase assays
β-Galactosidase activities of E. coli strains carrying bgaB-fusions (pBO1312, 1310, 1311,
1316, 1314, 1315, 1313, 602 and 1056 Table S1) were measured as described previously
(38), except that enzyme activity was measured at 55°C.
GFP-fluorescence assays
25 ml of LB-Cm medium was inoculated with 2 ml of E. coli DH5αZ1 overnight cultures,
carrying translational gfp-fusions (pBO1325, 1801 and 1802, Table S1) of the respective
hsp17 thermometer variants. Cultures were grown at 28°C to a final OD600 of 0.5 to 0.6. 10
ml of the culture was transferred to a prewarmed flask at 42°C. Subsequent to heat shock,
cells were shifted to 28°C for 90 min to allow full maturation of GFP-fusion proteins. Aliquots
were taken for Western analysis. Two times 2.5 ml of cells were spun down (13.000 rpm, 1
min) and resuspended in 500 µl 1x PBS buffer. 10 µl of cell suspension was pipetted on
D. Translation on demand by a simple RNA-based thermosensor
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(2)
9
object slides and fixed with 10 µl 0.5% low-melting agarose. The samples were analyzed
under a fluorescence microscope (BX51; Olympus, Hamburg, Germany).
Engineered Synechocystis strains
The Synechocystis strains used in this work were generated by transforming pNaive- hsp17
derivates (pBO1834, 1806 and 1807) into the kanamycin-resistant hsp17 deletion strain
HK1-1 (Kosaka and Fukuzawa, Kyoto University, Japan) kindly provided by Prof. Dr.
Elisabeth Vierling (University of Arizona). Transformations were performed according to
Klinkert et al. (33), prior to selecting for increasing spectinomycin resistance, at
concentrations up to 250 µg/ml spectinomycin dihydrochloride as described (29).
Homologous recombination of the hsp17 variants (WT, rep and derep) into Synechocystis
genome was confirmed by PCR, Northern and Western analysis.
Synechocystis heat shock assays
Heat shock treatments (42°C) for subsequent analysis were conducted by shifting cells from
28°C to prewarmed flasks at 42°C for various time lengths as indicated in the figure legends.
Synechocystis high light stress assays
High light experiments were performed by exposing cells to 600 µmol of photons m-2s-1 at
28°C for various time lenghts as indicated in the figure legends.
Chlorophyll determination
Chlorophyll determinations of Synechocystis cells was based on the method described by
Porra et al. (39). 500 µl of each culture was pelleted and resuspended in 1 ml MeOH prior to
sonification for 15 minutes. After centrifugation, absorbance of the supernatant was read at
652, 665.2 and 750 nm on a Novaspec spectrophotometer (Pharmacia Biotech, Freiburg,
Germany). Chlorophyll a (Chl a) concentration was calculated using the following formula:
Concentration (Chl a) [mg/ml] = 18.22 x (OD665.2 – OD750) – 9.55 x (OD652 – OD750)
D. Translation on demand by a simple RNA-based thermosensor
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Thylakoid stability assays
In vivo examinations of thylakoid thermostability were conducted with a luminescence
spectrometer (Aminco Bowman II, Thermo Fisher Scientific, Langenselbold, Germany) as
described (12). The temperature of respective Synechocystis cultures („WT“, Rep and
Derep) was increased at 2°C/min. Thylakoid photostability was determined by exposing cells
to high light at 28°C for 380 min. Changes in chlorophyll a (Chl a) fluorescence were
measured every 20 min.
Photosynthetic assays
A water-cooled Clark-type electrode (Bachofer, Reutlingen, Germany) was used to measure
the photosynthetic rates of Synechocystis strains carrying different hsp17 mutations.
Samples of heat shocked or high light stressed cells were immediately transfered to an
oxygen electrode chamber. To prevent CO2 depletion, 1mM NaHCO3 was added to the
culture as described (3). For analysis of cellular recovery, cells were transfered to
physiological conditions (28°C and low light).
In silico RNA structure prediction
RNA secondary structures were predicted by using the mfold server (http://
frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/rna-form1.cgi) running version 3.2 (40).
D. Translation on demand by a simple RNA-based thermosensor
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Results
Characterization of the first cyanobacterial RNA thermometer
In contrast to other RNA thermometers, the 5’-UTR of Synechocystis hsp17 exhibits a rather
minimalistic architecture. Based on the previously determined transcription start site (41,42),
the calculated structure of the 5’-UTR consists of a single hairpin of 44 nucleotides, in which
the SD sequence site is flanked by two loops, the internal asymmetric L1 loop and the large
L2 loop at the top (Fig. 1A). Nearly perfect canonical base pairing exists between the SD
(AGGAG) and the anti-SD (UCCUU) sequence. The AUG triplet is only partially embedded in
the structure at the base of the hairpin. The calculated free energy of the entire structure is
-5.5 kcal mol-1 (mfold; (40)).
A well-established Escherichia coli reporter gene system (27) was used to test
whether the cyanobacterial RNA element is able to confer temperature-dependent
translational control. The hsp17 5’-UTR element was cloned between the L-arabinose-
inducible pBAD promoter and the bgaB gene coding for a heat-stable β-galactosidase (43)
(Fig. 1B). At 28°C the wildtype (WT) fusion allowed a basal β-galactosidase activity of 30 MU
that increased 6-fold to 180 MU after a shift to 42°C (Fig. 1C). The induction by the hsp17 5’-
UTR is comparable to the Salmonella agsA fourU thermometer in our control experiments
and clearly exceeds induction of a control fusion to the 5’-UTR of the E. coli housekeeping
gene gyrA coding for the DNA gyrase.
As the efficiency of translation initiation correlates with the accessibility of the
ribosome binding site (44,45), stabilizing and destabilizing point mutations should have an
impact on RNA thermometer function. Selected sites for oligonucleotide-directed
mutagenesis are outlined in Fig. 1A. The first set of mutations (M1 to M4) was aimed at
stabilizing the overall thermometer structure. Indeed, M1, M2 and M3 significantly lowered
expression both at 28°C and at 42°C (Fig. 1C). Introduction of a G-C pair instead of the
kinked internal loop in M1 (AAC39-41G; henceforth called rep for “repressed”) resulted in
complete loss of reporter activity at both low and high temperatures. Mutation M2 (U17C)
D. Translation on demand by a simple RNA-based thermosensor
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A
C
B
D
D. Translation on demand by a simple RNA-based thermosensor
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Figure 1. Translational control by the hsp17 UTR element in E. coli. (A) The secondary structure as predicted by the mfold program (40) of the entire hsp17 5’-UTR is shown. The start codon (AUG,
marked by gray box) is located 45 nucleotides downstream of the transcription start site. The SD and anti-SD sequences, loop1 (L1) and loop2 (L2) are labelled. Site-directed mutations M1 to M4 and the
exchanged nucleotides are denoted in red letters; RR, variable nucleotides derived from random mutagenesis (primer: hsp17therm-M4-fw + hsp17therm-M4-rv, Table S1). (B) Schematic
representation of the reporter gene fusion on plasmid pBAD-bgaB. Additional nucleotides inserted due to the position of the NheI cloning site relative to the pBAD promoter are indicated by a box upstream
of the 5’-UTR. The artifical nucleotides at the 5’ end of the hsp17 transcript do not influence RNA folding and expression of the gene (data not shown). (C) Expression of the translational bgaB reporter
fusions (Miller Units, MU) to various hsp17 5’-UTRs. E. coli DH5α cells containing the corresponding
plasmids were grown in LB medium at 28°C and either kept at this temperature (white columns) or transferred to 42°C (black columns) for 30 min before β-galactosidase activity was measured. All
experiments were repeated at least in triplicate. Induction rates are shown above each fusion. A Salmonella agsA-bgaB fusion (fourU element; (27) was used as a positive control (C.1), while E. coli
13
substituted a G-U base pair by a more stable G-C base pair resulting in tighter repression at
both temperatures. M3 (U22G) was constructed to reduce the size of loop L2 (Fig. 1A). The
extra G-C pair provided additional stability to the stem and reduced β-galactosidase activity
at 28°C and 42°C but retained a 5-fold induction (Fig. 1C).
A collection of destabilizing mutations was obtained by partially randomized
oligonucleotides that introduced various nucleotides at the positions C19 and C20 (see
Material and Methods and supplementary Table S1). Four different hsp17 5’-UTR variants
(M4.1-M4.4) that were predicted to reduce the thermodynamic stability of the SD/anti-SD
interaction resulted in elevated expression at 28°C and 42°C (Fig. 1C). As an example, the
M4.4 variant (henceforth called derep for “derepressed”) produced 200 and 790 MU (as
compared to 30 and 180 MU, respectively, in the WT). Elevated reporter gene activity at
28°C resulted in reduced heat induction (2.5 to 3.9-fold) by all four constructs.
To demonstrate that the changes in expression levels at different temperatures were
due to translational control rather than to different transcript levels, we conducted Northern
blot experiments (Fig. 1D). The amounts of WT, rep and derep mRNAs were monitored at
28°C and 42°C using a probe directed against the coding region of the bgaB reporter gene.
Temperature changes had no influence on the steady-state level of the transcripts.
Translational control by the hsp17 5’-UTR was further assessed by a newly
developed green-fluorescent protein (gfp)-based reporter system (Fig. 2A). In contrast to the
bgaB system, it does not add any vector-derived nucleotides to the 5’ end of the transcript.
Transcription is driven by the PLtetO-1 promoter and initiates at the natural +1 site immediately
at the 5’ end of the inserted fragment (46). In E. coli DH5αZ1, this promoter is tightly
repressed by the Tet repressor and can be regulated by supplying 50 ng/ml doxycycline to
the culture. Western analysis demonstrated doxycycline-dependent and temperature-
D. Translation on demand by a simple RNA-based thermosensor
13
gyrA-bgaB (27) served as a negative control (C.2). Absolute β-galactosidase levels are listed in Table S2. (D) mRNA levels of hsp17-bgaB fusions before and after heat shock. Total RNA was extracted
from E. coli cultures incubated at either 28 or 42°C. Equal amounts were separated on a 1.2% denaturing agarose gel and Northern blot experiments were carried out using digoxygenin-labeled
RNA probes to detect bgaB transcripts. Ethidum-bromide stained rRNAs from the gel before blotting are shown as loading control. The bgaB fusion transcript runs at 2 kb.
14
controlled expression of the translational WT fusion (Fig. 2B). In case of the rep fusion,
expression occurred neither at 28°C nor at 42°C confirming the importance of loop L1 for
thermometer function. The derep fusion produced equal amounts of GFP protein at both
A
B
C
Figure 2. The hsp17 5’-UTR controls gfp expression in E. coli. (A) Schematic representation of the reporter gene fusions on the low-copy vector pXG10 (31). (B) Detection of GFP fusion proteins. E.
coli DH5αZ1 cells (46) carrying reporter plasmids with the WT, rep and derep hsp17 UTR element
were grown in LB medium at 28°C. Following heat shock as described in the legend to Fig. 1C, cells were incubated at room temperature for 90 min to allow fully maturation of GFP fusion proteins.
Samples were subjected to Western blot analysis using monoclonal α-GFP antibodies. Transcription
of the gfp fusions was induced by application of doxycycline (50 ng/ml), which inactivates the Tet
repressor. (C) Aliquots from the same samples were inspected by phase contrast and fluorescence microscopy. GFP fluorescence was excited at 460 nm, and light emission was recorded using a 510
nm filter. Representative examples are shown from three independent experiments, all of which gave similar results.
D. Translation on demand by a simple RNA-based thermosensor
14
15
28°C and 42°C. The determined protein levels were fully reflected by GFP fluorescence in
vivo (Fig. 2C). Fluorescence microscopy revealed weak basal GFP activity at 28°C from the
WT fusion that was enhanced when cells were shifted to 42°C. The rep variant produced no
fluorescence at all, whereas cells containing the derep fusion emitted fluorescence at both
temperatures.
Taken together, the 5’-UTR of the Synechocystis hsp17 gene displays all features
characteristic of a typical RNA thermometer when tested in E. coli reporter systems. The rep
and derep versions are presumably locked into “closed” and “open” states, respectively.
Temperature-mediated structural changes in the hsp17 leader sequence
To map the structure and temperature responsiveness of the hsp17 5’-UTR in vitro, we
synthesized WT, rep and derep fragments by T7 RNA polymerase-mediated in vitro
transcription and performed comparative enzymatic and chemical probing experiments.
Cleavages introduced at 28 and 42°C by lowest enzyme or lead(II) concentrations,
respectively, were used for structural interpretations. The cleavage pattern of the WT UTR
after limited digestion with various RNases indicated that the computer-predicted structure
(Fig. 1A) is almost correct (Fig. 3A). Nucleotide G9 proposed to mask the uridine of the AUG
start codon appeared to be partially single-stranded at 28°C and became highly susceptible
to RNase T1, which cuts 3’ of single-stranded nucleotides, at 42°C. Temperature-mediated
disengagement of the AUG start codon was also suggested by enhanced T1 cleavage of
G47 at 42°C. The SD sequence-forming nucleotides G33, G34 and G36 were moderately
cleaved by RNase T1 at 28°C. The susceptibility of this region was significantly increased at
42°C, providing evidence for temperature-induced liberation of the SD region. G36 was most
sensitive towards RNase T1 suggesting that melting initiates from L1 towards G36.
The structure of the rep RNA clearly deviates from the WT structure (Fig. 3A). Absent
T1 cuts supported the predicted tight structure. Nucleotides U42 and A43 were cleaved by
RNase V at low and high temperature suggesting permanent sequestration of the start
codon. RNase V primarily cleaves double-stranded and stacked regions. The anti-SD
nucleotides were also cleaved by RNase V at 28 and 42°C. Most importantly, the SD
D. Translation on demand by a simple RNA-based thermosensor
15
16
sequence did not become accessible to RNase T1 at 42°C indicating a “closed” state, in
which the SD sequence is not accessible to the 16S rRNA.
Figure 3. Enzymatic and chemical probing of hsp17 UTR variants. (A-B) Enzymatic hydrolysis and lead(II)-induced cleavages performed on 5’-end-labeled hsp17 5’-UTR at 28°C or 42°C. The
conditions for RNase and lead(II) concentrations were as follows: (A) RNase T1 (0.004U and 0.01U), RNase V (0.008U and 0.02U); (B) lead(II) (10 and 20 mM). RNA fragments were separated on 8%
polyacrylamide gels. Lanes N, controls without RNase or modifying agents; lanes T, RNase T1 cleavages under denaturing conditions; lanes L, alkaline ladder. SC (Start Codon), L1 (internal
Loop1), SD (Shine Dalgarno), L2 (Loop2) and anti-SD regions are indicated. (C) Computer-predicted secondary structures and enzymatic and lead(II)-induced cleavage sites of WT and mutated hsp17
UTRs. Cleavage sites introduced at 28°C by lowest enzyme or lead(II) concentrations are depicted by arrows as indicated. Circled nucleotides are highly susceptible to RNase T1 at 42°C. Enhanced
lead(II) cleavage at 28°C occured at nucleotides marked by asterisks.
A B
C
D. Translation on demand by a simple RNA-based thermosensor
16
17
In the derep RNA, the introduction of two Gs instead of two Cs at position 19 and 20
(pairing with the two Gs of the SD sequence in the WT RNA, Fig. 1A) resulted in an altered
and less stable structure with a shifted L2 loop and a wider L1 loop (Fig. 3C). The instable
structure at low temperature was most evident by chemical probing (Fig. 3B). Lead was used
as a single-strand-specific probe to detect subtle conformational changes in rather flexible
regions, e.g. internal loops and bulged nucleotides (47). Nine nucleotides (A12, U13, C14,
U44, and in particular A15, A16, C41, A42, U43) in or flanking the internal loop L1 of the
derep RNA were attacked by lead (II) at 28°C. Loop L1 of the WT structure was much less
sensitive towards lead(II). Lead probing confirmed the predicted existence of the L2 loop in
the WT and rep RNAs. The SD sites of all RNAs remained unaffected by lead(II) attack
probably due to strong base stacking of the purine nucleotides.
Collectively, these results support the model that the 5’-UTR of Synechocystis hsp17
blocks access to the SD sequence at low temperatures and responds to a temperature
upshift by melting that liberates the ribosome binding site.
To provide further evidence for the melting process and the differences between the
three RNA species, commercially synthesized RNA was subjected to temperature-gradient
circular dichroism (CD) spectrometry (Fig. 4A). The unfolding curves exhibit Tm values of
45.1°C for the derep RNA, 50.2°C for the WT RNA and 56.9°C for the rep RNA. Sigmoidal
Figure 4. Melting studies of hsp17 thermometer element by CD spectroscopy. (A) Temperature-dependent fraction of unfolded RNA α(T) of the hsp17 WT (blue line), derep (black line) and rep (red
line) RNA as derived from CD unfolding curves recorded at a wavelength of 263 nm. (B) Comparison
of unfolding and refolding α(T) curves of the hsp17 WT RNA as derived from CD unfolding and
refolding curves recorded at a wavelength of 263 nm.
A B
D. Translation on demand by a simple RNA-based thermosensor
17
18
fitting of the α(T) curves results in Hill coefficients of b = 6.1 for the derep RNA, b = 4.2 for
the WT RNA and b = 8.2 for the rep RNA. Unfolding and refolding curves of the WT RNA are
nearly identical (Fig. 4B) indicating that unfolding is a completely reversible process.
Temperature-controlled ribosome binding to the hsp17 thermometer
In order to directly examine binding of the ribosome to the Synechocystis hsp17 5’-UTR, we
performed toeprinting (primer extension inhibition) assays. Complex formation between the
radio-labelled primer, 30S ribosome and initiator tRNA and the hsp17 UTRs (WT and derep:
104 nucleotides; rep: 102 nucleotides; see full length products in Fig. 5) was allowed at 28°C
or 42°C. The extent of ternary complex formation was determined by primer extension.
Prematurely terminated products (toeprints) corresponding to the +17 position downstream
Figure 5. Temperature-dependent ribosome binding to the hsp17 5’-UTR. Toeprinting was carried out on 2 pmol of WT, rep and derep RNAs as described in Materials and Methods. The absence (-) or
presence (+) of 30S subunits is indicated. Terminated reverse transcription products (toeprints) at position +17 relative to the A of the translation start codon and full-length products are pointed out by
arrows. TGCA refers to a sequencing ladder generated with the same oligonucleotide as in the toeprint experiments. The position of the start codon is boxed.
D. Translation on demand by a simple RNA-based thermosensor
18
19
of the AUG start codon were expected if ribosome was bound to the RNA. At 28°C, the
majority of reverse transcripts were extended to the 5’ end of the template (Fig. 5). Only
small amounts of truncated cDNAs were obtained at the characteristic toeprint position. In
contrast, prominent toeprint signals accompanied by a reduced yield of full-length cDNA
occurred at 42°C indicating that formation of a translation initiation complex was greatly
stimulated at heat shock temperatures.
The complete absence of a toeprint signal when the rep RNA was used and
constitutive, temperature-independent formation of a ternary complex with the derep RNA
confirmed the importance of the RNA architecture for ribosome binding.
Introduction of hsp17 thermometer variants into the Synechocystis background
As reported previously, inactivation of the Synechocystis hsp17 gene results in reduced
thermotolerance and thylakoid stability under heat stress conditions (3,48). Conversely,
constitutive expression of hsp17 confers thermotolerance and protection of the
photosynthetic apparatus (49). This offers the unique opportunity to test for the physiological
importance of the hsp17 thermometer element. We made use of a Δhsp17 mutant and
engineered stable Synechocystis strains carrying chromosomally integrated WT, rep and
derep 5’-UTRs upstream of hsp17 (henceforth called „WT“, Rep and Derep strains). The
Synechocystis HK-1 strain, which lacks the hsp17 open reading frame sll1514 (50) was used
for homologous recombination via flanking sequences enabling selection for increasing
spectinomycin resistance (Fig. 6A). Correct insertion into the Synechocystis genome was
confirmed by PCR (data not shown).
Expression of hsp17 in the recombinant strains was assayed by Northern and
Western blot analyses. All three strains produced comparable amounts of hsp17 mRNA (Fig.
6B and C). Consistent with previous results (42), the level of hsp17 transcript was low at
28°C and increased after a shift to 42°C for 1 hour (Fig. 6B). Despite the presence of hsp17
mRNA, the small heat shock protein was not detectable at low temperature suggesting
translation inhibition. Hsp17 protein accumulated at 42°C. The Rep strain was incapable of
translating hsp17 mRNA whereas the Derep mutant synthesized equal amounts of Hsp17 at
28°C and at 42°C.
D. Translation on demand by a simple RNA-based thermosensor
19
20
Induction of hsp17 in Synechocystis occurs not only after heat-shock but also after
exposure to strong visible light (8). As Synechocystis cells exposed to 300 µmol photons
m-2s-1 transcribe only little hsp17 mRNA (3), we choose higher light intensities of 600 µmol
photons m-2s-1 for 30 min. Light stress-dependent synthesis of Hsp17 in „WT“, Rep and
Derep cells followed the same pattern as in heat-shocked cells; i.e. it was induced, absent or
constitutively high, respectively (Fig. 6C). Comparative Western blot analysis showed that
heat shock is a stronger inductor of hsp17 expression than high light stress (Fig. S2).
Moreover, Hsp17 protein is a very stable protein as it was still traceable in the late recovery
phase (60 min post-shock) of stressed „WT”, Rep and Derep strains.
Figure 6. Effect of chromosomally integrated hsp17 UTR variants on Hsp17 production in Synechocystis. (A) Outline of the strategy for construction of hsp17 mutants. The Synechocystis
HK-1 (Δhsp17) strain was used for integration of hsp17 and its 5’-UTR via upstream (red) and
downstream (blue) flanking sequences. SmR, spectinomycin resistance; KmR, kanamycin resistance. Homologous recombination resulted in generation of the so-called „WT“, Rep and Derep strains. (B and C) Determination of hsp17 mRNA and Hsp17 protein levels by Northern and Western analysis, respectively. (B) Total RNA and protein was extracted from Synechocystis cells incubated at either 28
or 42°C. Ethidium bromide stained 16s rRNA served as a loading control in Northern blot experiments. Equal amounts of total protein were checked by Coomassie-stained SDS-PAGE gels
(data not shown). A digoxygenin-labelled RNA probe was used to detect hsp17 transcripts, Hsp17 protein was detected via monoclonal α-Hsp17 antibody. (C) Synechocystis cells were incubated under
low light (LL: 30 µmol photons m-2s-1) or high light (HL: 600 µmol photons m-2s-1) conditions at 28°C
for 30 min. Temperature-stability of the culture was monitored. Subsequent Northern and Western analysis was conducted as described above.
A
B C
D. Translation on demand by a simple RNA-based thermosensor
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21
Open and closed hsp17 UTRs confer growth defects under stress conditions
In order to determine their stress resistance, Synechocystis „WT”, Rep and Derep strains
were challenged with different stress conditions. First, cells were exposed to a 6 h heat
shock at 42°C and subsequently incubated at 28°C under low light conditions (30 µmol
photons m-2s-1) for 5 days (Fig. 7A, top panel). The pale phenotype showed that the Δhsp17
strain was sensitive to this treatment. The complemented „WT” strain was protected, and the
Rep and Derep mutants behaved like the Δhsp17 and „WT” strain, respectively.
Heat sensitivity of the Δhsp17 and Rep strains coincided with a reduced Chlorophyll a
(Chl a) content (1.0 mg/ml in Δhsp17 and 1.5 mg/ml in Rep compared to 6.0 mg/ml in „WT”
cells; Fig. 7B). As Chl a is functionally linked to PSII reaction centers, the reduced Chl a
concentrations suggest a defective photosynthetic apparatus in the absence of Hsp17. When
the four strains were exposed to high light (600 µmol photons m-2s-1) for 6 h at 28°C, they
displayed a similar phenotype as after heat stress (Fig. 7A, second panel). However, light
stress caused less Chl a decay in Δhsp17 and Rep cells (3-fold and 2-fold, respectively; Fig.
7B) indicating a milder impact on cellular fitness. A defect of the Derep mutant became
evident when a combined heat/light stress was applied (Fig. 7A, third panel) suggesting that
permanent overproduction of Hsp17 alters cell physiology. Here, a heat shock for 1 h was
applied prior to 5 h high light exposure. While the „WT” strain was protected against this
severe stress, the Δhsp17 and the Rep strain were equally sensitive. The Derep mutant
showed an intermediate phenotype retaining one third of its original Chl a content (Fig. 7B).
The importance of appropriate amounts of Hsp17 for the fitness of Synechocystis was
further demonstrated by growth tests in the presence (heterotrophic growth) or absence
(phototrophic growth) of glucose. The Rep mutant had a strongly reduced growth rate
compared to the „WT” under all stress conditions tested (Fig. 7C). The Derep strain grew well
heterotrophically in the presence of glucose. Only under combined heat/light stress
conditions the growth rate was reduced by 10%. Under photosynthetic growth conditions in
the absence of glucose, however, the Derep strain suffered from continuous Hsp17
production. The growth rate was reduced to 60 % under light stress and to 40% under heat/
D. Translation on demand by a simple RNA-based thermosensor
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22
A
B
C
D. Translation on demand by a simple RNA-based thermosensor
22
Figure 7. Stress-induced phenotypes of Synechocystis hsp17 mutants. (A) Cells were grown to early log phase prior to stress treatment. HS, cells were incubated at 42°C for 6 h; HL, cells were
exposed to 600 µmol photons m-2s-1 for 6 h; HL*, cells were incubated at 42°C for 60 min prior to 5 h
HL (600 µmol photons m-2s-1) treatment. Following the different stress conditions, the cultures were
transferred to 28°C and LL conditions for 5 days. Before documentation, cultures were diluted to an
23
light stress suggesting that deregulated hsp17 expression in particular affects the
photosynthetic apparatus.
Open and closed hsp17 thermometer elements affect photosynthesis activity of
Synechocystis
The Hsp17 protein stabilizes the lipid phase of membranes and contributes to the
maintenance of the thylakoid integrity, especially under stress conditions (7). This prompted
us to examine the integrity of the photosynthetic apparatus in our hsp17 mutants. Chlorophyll
a (Chl a) fluorescence can be used as an intrinsic probe for the stability of PSII-protein
complexes (12). Loss of thylakoid membrane stability is correlated with an increase in Chl a
fluorescence. Maximal fluorescence in the „WT” strain was measured at 48°C (Fig. 8A). A
striking reduction of thermal stability to 42°C was monitored in the Rep mutant whereas
thermostability of the photosynthetic apparatus was increased to 55°C in the Derep strain.
Light stress led to comparable results (Fig. 8B). Maximal Chl a fluorescence was measured
in the „WT” after 200 min of high light exposure. Photostability was reduced to 140 min in the
Rep mutant and increased to 300 min in the Derep strain.
To measure photosynthetic activity of the hsp17 mutants, we determined oxygen
evolution after exposure to 42°C or high light conditions. While there was a drop in oxygen
evolution of about 30% during the first hour at 42°C in the „WT”, the Derep mutant was
barely affected suggesting that the photosynthetic machinery was well-protected (Fig. 8C).
Heat sensitivity of the Rep mutant on the other hand was reflected by a sharp decrease in
oxygen evolution to 20 % within 60 min after heat shock. Interesting differences were
observed in the recovery phase after the cultures had been returned to 28°C and low light
conditions. The „WT” and in particular the Rep strain, which is unable to produce Hsp17,
D. Translation on demand by a simple RNA-based thermosensor
23
optical density (OD730) of 1.5. Representative examples of at least three experimental repetitions are shown. (B) Samples from these Synechocystis cell cultures were used for Chl a extraction. (C) Early
log phase cells grown in the presence or absence of glucose were treated with HS, HL, HL* as described in (A). For growth rate (µ) determination, cells were transferred to 28°C and LL conditions
for 48 h (µ= lg2*OD-lg2*OD0/t-t0). Growth of the Rep and Derep mutants relative to the „WT” cultures is
shown.
24
recovered rapidly and regained full oxygen evolution capacity within one hour. In contrast,
the Derep mutant suffered most in the recovery phase and returned to pre-shock efficiency
only slowly indicating that continued production of Hsp17 is counterproductive once the
stress has declined.
Figure 8. Integrity of the photosynthetic machinery in Synechocystis strains with mutated hsp17 5’-UTRs. (A) The temperature effect on Chl a fluorescence was measured over a temperature
range from 30 to 60°C in „WT“ (circles), Rep (squares) and Derep (triangles) cells grown at 28°C under LL conditions. (B) The impact of light stress on Chl a fluorescence was measured in cells from
the same pre-culture as in (A). Cultures were exposed to HL over period of 380 min. (C) Oxygen evolution was monitored after a heat shock from 28 to 42°C at the indicated time points. After 380
min, cultures were returned to 28°C. (D) Oxygen evolution after shift to HL conditions. For recovery, the cultures were returned to LL conditions for 60 min. Cells from the same cultures were used for the
experiments in (C) and (D). Results are presented as percentage of the oxygen evolution rate measured at time 0. The photosynthetic activities before stress treatment were 3.02±0.04, 2.96±0.01
and 2.87±0.07 µmol O2 mg Chl-1min-1 in the „WT“ (circles), Rep (squares) and Derep (triangles)
stains, respectively. Error bars represent standard deviations obtained from three independent experiments.
A
B D
C
D. Translation on demand by a simple RNA-based thermosensor
24
25
Exposure to high light intensities had a similar effect on oxygen evolution. Heat-
preincubation of the cells increased the effect of high light stress in all strains (data not
shown). Again, intermediate damage after stress exposure occurred in the „WT” background
(Fig. 8D). The Rep mutant was most sensitive and the Derep strain was barely affected.
Under recovery conditions, the „WT” and Rep strain performed best, whereas the Derep
strain was delayed in full oxygen production. Apparently, well-adjusted synthesis of Hsp17
protein by the hsp17 UTR is important for stress management in Synechocystis.
D. Translation on demand by a simple RNA-based thermosensor
25
26
Discussion
A novel RNA thermometer family
RNA thermometers are widely distributed genetic control elements in the 5’-UTR of
temperature controlled genes (23,51). They have been found in numerous α- and γ-
proteobacteria and in the Gram-positive pathogen Listeria monocytogenes (25,27,52). Here,
we document the first cyanobacterial RNA thermometer that controls translation initiation of
the hsp17 gene in Synechocystis sp. PCC 6803. Consisting of only 44 nucleotides forming a
single stemloop structure it is by far the shortest and most simple natural RNA thermosensor
known to date. Typical ROSE (Repression Of heat Shock gene Expression) elements are
between 60 and 110 nucleotides long (52). The fourU element upstream of the Salmonella
agsA gene contains 58 nucleotides (27). The E. coli rpoH thermometer that reaches far into
the coding region is composed of 227 nucleotides (24). In its simplicity, the cyanobacterial
element resembles rationally designed synthetic RNA thermometers consisting of a short
single stemloop (53).
Unfolding curves (Fig. 4) supported the different stabilities of the WT, derep and rep
RNAs observed by reporter gene fusions in vivo and probing results in vitro. According to the
CD measurements the WT RNA exhibits a higher Tm value than the destabilized derep RNA
and a lower Tm value than the stabilized rep RNA. Interestingly, the unfolding transition of the
WT RNA is extraordinarily uncooperative (b=4.2). The derep RNA shows slightly higher
cooperativity (b=6.1) and the unfolding transition of the rep RNA exhibits the highest
cooperativity (b=8.2). In comparison to the Salmonella fourU RNA thermometer (b=10.6)
(Rinnenthal et al., 2010) the cooperativity of the Synechocystis thermometer is very low
(b=4.2). In other words, the cyanobacterial RNA exhibits a more gradual dimmer-like
behavior than the Salmonella fourU element.
Riboswitches and RNA thermometers are simple regulatory elements that have
repeatedly been suggested to originate from an ancient RNA world (23,54,55). Recently
discovered cyanobacterial fossils were dated 3.5 billion years old (56) marking the advent of
oxygenic photosynthesis. Apparently the presence of a functional hsp17 5’-UTR is beneficial
D. Translation on demand by a simple RNA-based thermosensor
26
27
to Synechocystis sp. PCC 6803. This prompted us to ask whether other cyanobacteria
possess similar elements. Interestingly, likely candidates were identified in the long 5’-UTRs
of the Thermosynechococcus elongatus and T. vulcanus small heat shock gene hspA. At
their 3’ end they exhibit significant sequence similarity to the hsp17 leader sequence
(supplementary Fig. S1). According to in silico calculations the extended 5’ end is predicted
to form a long hairpin structure which might be required for overall stability of the RNA
structure in a thermophilic habitat. The presence of thermometer-like sequences upstream of
small heat shock genes in distantly related, thermophilic cyanobacteria suggests that this
translational control element might be commonly used to withstand constantly changing
temperature and light conditions.
Physiological relevance of the cyanobacterial thermometer
Our study provides first experimental evidence for the physiological importance of the on-
and off-function of an RNA thermometer. Although the structure and temperature-induced
conformational changes of several RNA thermometers have been studied in detail, evidence
for their in vivo significance has been lacking except for the Listeria prfA thermosensor that
activates virulence gene expression inducing host cell invasion (25). A major reason is that
most known RNA thermometers control translation of small heat shock genes, which usually
are dispensable because the cellular chaperone network is very redundant (5). In contrast,
the hsp17 gene is critically important for Synechocystis because its major target, the
photosynthetic machinery is very sensitive to stress-induced damage. Therefore, Hsp17 is
essential for tolerance to high temperatures and light stress in several mesophilic and
thermophilic cyanobacteria (3,49,57). Heterologous expression of the sHsp from
Synechococcus vulcanus in Synechococcus sp. PCC 942 conferred increased thermal
resistance and prevented inactivation of the photosynthetic apparatus (49). The protective
effect of the amphitrophic Hsp17 protein depends on its dual role as a protein and membrane
chaperone (7). In vitro, Hsp17 is able to protect model proteins from heat-induced
aggregation (7). In vivo, heat stress-induced Hsp17 interacts with a large variety of proteins
and protects a wide range of cellular functions (58). The second important activity is its
membrane association (59). Most newly synthesized Hsp17 is associated with the thylakoid
D. Translation on demand by a simple RNA-based thermosensor
27
28
membrane (12). Here it plays a critical role in controlling the physical order, bilayer stability
and integrity of membranes (7). Further evidence for the importance of this property derives
from an hsp17 mutant with increased thylakoid association, which provided elevated
resistance against UV stress (60). Isolated Synechococcus elongatus PCC7942 phycocyanin
interacts directly with HspA, a Hsp17 homolog (57). Thus, the severe defect of stressed
Synechocystis Δhsp17 and Rep mutants (Fig. 7A) might be due to detachment and
degradation of accessory pigments. This assumption is well supported by the reduced Chl a
levels (Fig. 7B).
Despite the importance of Hsp17 for photosynthetic activity, the mechanisms
controlling its expression were largely unexplored. Alternative sigma factors are known to be
required for transcription of hsp17. However, the mRNA is not translated in the absence of
stress (Fig. 6). Consistent with our Northern blot experiments, DNA microarray assays
revealed basal transcription of the hsp17 gene even if the inducing SigB factor was missing
(10,11). The absence of Hsp17 protein under normal growth conditions despite the presence
of significant amounts of hsp17 mRNA (Fig. 6) indicates that the 5’-UTR of hsp17 blocks
translation initiation (Fig. 9) when the chaperone is not required. In vitro ribosome binding
assays demonstrated that translational is strictly temperature-controlled and does not require
the aid of additional cellular factors. Constitutive basal transcription of hsp17 apparently
provides a cellular pool of mRNA intended for instantaneous „translation-on-demand“, in
case the conditions become unfavourable.
Under stress conditions, the simultaneous induction of hsp17 transcription and
translation allows rapid accumulation of Hsp17 protein (Fig. 9). In turn, the sHsp promotes
readjustment of membrane fluidity and repair of unfolded proteins in the cytoplasm. In the
post-stress phase, rapid shut-down of Hsp17 synthesis is achieved by the thermometer
inherent to the mRNA. The defect of the Derep mutant under phototrophic conditions and in
the stress recovery phase strongly suggests that turning off hsp17 translation is as important
as turning it on. Apparently, permanent accessibility of the SD sequence is deleterious in the
recovery phase when hsp17 mRNA levels are still very high (Fig. 9). Immediate
sequestration of the SD sequence ensures that the abundant mRNA is silenced after
restoration of physiological conditions.
D. Translation on demand by a simple RNA-based thermosensor
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29
In the course of our experiments we made the puzzling observation that translation of
hsp17 mRNA was not only induced by heat stress but also, although weaker (Fig. S2), by
high light conditions (Fig. 6). As we constantly monitored temperature during light exposure,
we exclude that the observed induction was simply due to heating-up the growth medium.
Thus, it appears that the hsp17 thermometer responds to high light stress. At this point we
can only speculate on the mechanism underlying the induction of hsp17 translation. The
exposure of phototrophic organisms to light quantities above the level required for saturating
photosynthetic electron flow results in irreversible damage to the D1 protein and inactivation
of PSII (61). To avoid progressive photoinactivation, excess photon energy is partially
Figure 9. The 5’-UTR of the Synechocystis hsp17 gene controls translation on demand. The WT RNA forms a secondary structure at physiological temperature (28°C) masking the ribosomal
binding site of hsp17, thus preventing the binding of the ribosome. A heat shock induces hsp17 transcription. Concomitant melting of the hairpin structure in the 5’-UTR allows translation initiation. A
downshift to physiological temperatures shuts off translation in spite of high transcript levels. Transcriptional induction of the Derep and Rep variants is unaffected. However, the blocked SD
sequence in the Rep mutant prevents translation under all conditions, whereas translation is constitutively on in the Derep mutant.
D. Translation on demand by a simple RNA-based thermosensor
29
30
dissipated as heat from non-functional PSII centres (62,63). Although this might not induce a
global heat shock response, it might be sufficient to cause partial melting of an RNA
thermometer given the prolonged and intensive high light stress (we were not able to induce
Hsp17 translation with visible light intensities below 600 µmol photons m-2s-1). In this context
it is interesting that de novo synthesis of thylakoid-associated Hsp17 at ambient temperature
can be induced by altering membrane microviscosity by addition of 30 mM benzyl alcohol
(12). As interaction of benzyl alcohol with ribonucleic acids has been reported, the alcohol
might be able to induce partial melting of an RNA thermometer (64). Ethanol also was shown
to induce sHsp biosynthesis controlled by the ROSE thermometer in Bradyrhizobium
japonicum (65). In any case, alcohol was a much weaker inducer of sHsp biosynthesis than
heat shock.
In γ-proteobacteria like E. coli and Salmonella, RNA thermometers modulate heat
shock gene expression by adding an additional layer of control to the efficient transcriptional
induction by the alternative sigma factor σ32 (27,36,66). Instead, the hsp17 thermometer is
the predominant regulator of hsp17 expression in Synechocystis. Severe phenotypes were
elicited by the exchange of only a few nucleotides in the chromosomally integrated
thermometer. Weakening the SD/anti-SD interaction by two point mutations resulted in
permanent hsp17 translation, which offered a significant protection to the photosynthetic
apparatus in the first hours after stress exposure. As a drawback, cells with elevated Hsp17
levels were delayed in stress recovery and showed reduced fitness under photosynthetic
growth conditions in the presence of stress. Apparently, the architecture of the hsp17 UTR is
well-adjusted to supply the perfect amount of the chaperone under normal, stress and
recovery conditions. It is a sensitive, efficient and rapidly responding gene control element
that performs equally well in vitro, in E. coli and in Synechocystis making it an attractive
candidate for applications in green biotechnology, which uses cyanobacteria or plants as
biofactories (67). Owing to its simple architecture the Synechocystis element is much more
suitable as versatile gene control element than all other previously described RNA
thermometers.
D. Translation on demand by a simple RNA-based thermosensor
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31
Funding
This work was funded by a grant from the German Research Foundation (DFG priority
program 1258) to FN and by a fellowship from the Studienstiftung des Deutschen Volkes to
JK.
Acknowledgements
We thank Elizabeth Vierling and Heather O’Neill (University of Arizona) for providing strains,
plasmids and α-Hsp17 antisera. We are grateful to Matthias Rögner, Thilo Rühle, Birgit
Klinkert, Nicole Frankenberg-Dinkel and Sebastian Rasche for experimental support and to
Ulla Aschke-Sonnenborn for technical assistance. All members of the RNA group, Nicole
Frankenberg-Dinkel and Bernd Masepohl are acknowledged for critical comments on the
manuscript and helpful discussions.
D. Translation on demand by a simple RNA-based thermosensor
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32
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Figure S1. Alignment of Synechocystis hsp17 5’-UTR (thermometer element) with the 5’-UTRs of T. elongatus and T. vulcanus. Identical nucleotides are indicated by bold letters.
Figure S2. Comparative Western blot analysis of Hsp17 protein levels after heat shock (HS) and high light stress (HL) and in the recovery phase. (A) Synechocystis „WT“ and (B) Derep cells were incubated at 42°C (for 60 min) or stressed with high light (for 30 min). Following the different stress
conditions, the cultures were transferred to 28°C and LL conditions (recovery phase). Total protein was extracted directly after stress (HS or HL) and at time points during recovery phase as indicated. K,
control (total protein extracted from cells grown at 28°C and low light). (C) Total protein was extracted from Synechocystis Rep cells incubated at 42°C or under high light conditions and blotted beside total
protein originating from heat shocked „WT“ cells. Hsp17 protein was detected via monoclonal α-Hsp17 antibody. Equal amounts of total protein were checked by Coomassie-stained SDS-PAGE gels (data
not shown).
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Table SI. Strains, plasmids and oligonucleotides used in this study
Strain, plasmid or oligonucleotide
Relevant characteristic(s) or sequencea Source or reference
StrainsE. coli DH5α supE44 ΔlacU169 (Φ80lacZΔM15) hsdR17 recA1 gyrA96 Gibco-BRLE. coli DH5αZ1 Δ(argF-lac)I69ϕ 80ΔlacZ58(M15) glnV44 (AS) rfbD1 gyrA96 (NalR) recA1
endA1 sρoT1 thi-1hsdRI7 ZI (lacR tetR SpR)Lutz and Bujard, 1997
Synechocystis sp.
PCC6803 HK-1
Δ hsp17 (KmR) Kosaka and Fukuzawa
PlasmidspBAD-bgaB
(pBO415)
Translational bgaB fusion vector, bgaB: heat-stable β-galactosidase, AmpRWaldminghaus et al., 2007
pXG-10 Translational gfp fusion vector, CmR, PLTet-O Promotor Urban and Vogel, 2006pNaive.16 (pAZ877) Vector for integration into the hsp17 locus, Restriction sites used for
subcloning: BamHI (upstream region); CpoI (hsp17 coding region), SpecR , AmpR
Giese and Vierling, 2002
pBO1292 Synechocystis hsp17 5’-UTR (thermometer element) in pUC18 (QuikChange® template)
This study
pBO1347 fragment of ORF sll1514 containing hsp17 thermometer element in pUC18, (pNaive-QuikChange® template)
This study
pBO1293 Synechocystis hsp17 5’-UTR-bgaB fusion in pBAD-bgaB This studypBO1312 Synechocystis hsp17 5’-UTR-AAC39-41G-bgaB fusion in pBAD-bgaB
(M1-rep)This study
pBO1310 Synechocystis hsp17 5’-UTR-T17C-bgaB fusion in pBAD-bgaB (M2) This studypBO1311 Synechocystis hsp17 5’-UTR-T22G-bgaB fusion in pBAD-bgaB (M3) This studypBO1316 Synechocystis hsp17 5’-UTR-C19T-bgaB fusion in pBAD-bgaB (M4.1) This studypBO1314 Synechocystis hsp17 5’-UTR-CC19/20GA-bgaB fusion in pBAD-bgaB
(M4.2)This study
pBO1315 Synechocystis hsp17 5’-UTR-CC19/20AG-bgaB fusion in pBAD-bgaB (M4.3)
This study
pBO1313 Synechocystis hsp17 5’-UTR-CC19/20GG-bgaB fusion in pBAD-bgaB (M4.4-derep)
This study
pBO602 Salmonella agsA-bgaB fusion in pBAD-bgaB (Control C.1) Waldminghaus et al., 2007
pBO1056 E. coli gyrA-bgaB fusion in pBAD-bgaB (Control C.2) Waldminghaus et al., 2007
pBO1325 Synechocystis hsp17 5’-UTR-gfp fusion in pXG-10 This studypBO1801 Synechocystis hsp17 5’-UTR-rep-gfp fusion in pXG-10 This studypBO1802 Synechocystis hsp17 5’-UTR-derep-gfp fusion in pXG-10 This studypBO1301 Synechocystis hsp17 5’-UTR-WT-in vitro transcripts for structure probing
experiments, runoff vector (via MlsI site), pUC18-basedThis study
pBO1304 Synechocystis hsp17 5’-UTR-rep-in vitro transcripts for structure probing experiments, runoff vector (via MlsI site), pUC18-based
This study
pBO1302 Synechocystis hsp17 5’-UTR-derep-in vitro transcripts for structure probing experiments, runoff vector (via MlsI site), pUC18-based
This study
pBO1305 Synechocystis hsp17 5’-UTR-WT-in vitro transcripts (+63 bp coding region) for toeprinting experiments, runoff vector (via HpyCH4V site), pUC18-based
This study
pBO1349 Synechocystis hsp17 5’-UTR-rep-in vitro transcripts (+63 bp coding region) for toeprinting experiments, runoff vector (via HpyCH4V site), pUC18-based
This study
pBO1348 Synechocystis hsp17 5’-UTR-derep-in vitro transcripts (+63 bp coding region) for toeprinting experiments, runoff vector (via HpyCH4V site), pUC18-based
This study
pB01834 pNaive- hsp17 5’-UTR-WT (pNaive.16 derivate) This study
pBO1806 pNaive- hsp17 5’-UTR-rep (pNaive.16 derivate) This study
pBO1807 pNaive- hsp17 5’-UTR-derep (pNaive.16 derivate) This study
OligonucleotidesNheI-hsp17therm TTTGCTAGCATTCAAGGGTAATCAA
(*pBO1293)hsp17therm-EcoRI TTTGAATTCAGACATAATGTTAACTCC
(*pBO1293)hsp17therm-M1-fw CACACATCAGGAGTTGATTATGTCTGAATTC
(*pBO1312, pBO1801, pBO1304 and pBO1806)hsp17therm-M1-rv CTCCTGATGTGTGGCAGGAATTGATTACCC
(*pBO1312, pBO1801, pBO1304 and pBO1806)
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39
hsp17therm-M2-fw CAAGGGTAATCAACTCCTTCCACACATCAGG
(*pBO1310)hsp17therm-M2-rv CCTGATGTGTGGAAGGAGTTGATTACCCTTG
(*pBO1310)hsp17therm-M3-fw GGGTAATCAATTCCTGCCACACATCAGGAG
(*pBO1311)hsp17therm-M3-rv CTCCTGATGTGTGGCAGGAATTGATTACCC
(*pBO1311)hsp17therm-M4-fw CAAGGGTAATCAATTRRTTCCACACATCAGG
(*pBO1316, pBO1314, pBO1315 and pBO1313)hsp17therm-M4-rv CCTGATGTGTGGAARRAATTGATTACCCTTG
(*pBO1316, pBO1314, pBO1315 and pBO1313)hsp17therm-der.-fw CAAGGGTAATCAATTGGTTCCACACATCAGG
(*pBO1802, pBO1302 and pBO1807)hsp17therm-der.-rv CCTGATGTGTGGAACCAATTGATTACCCTTG
(*pBO1802, pBO1302 and pBO1807)PstI-rep-fw TTTCTGCAGATTCAAGGGTAATCAATTGG
(*pBO1802)derep-NheI-rv AAAAGCTAGCAGACATAATCAACTCCTG
(*pBO1801)T7- hsp17therm GAAATTAATACGACTCACTATAGGGATTCAAGGGTAATCAATTCC
(*pBO1301 and pBO1305)hsp17therm-runoff-SP TGGCCAGACATAATGTTAACTCCTG
(*pBO1301)hsp17 therm-runoff-toeprinting
TGCAAACAGTTGGTTCATCTGCTGCTGG
(*pBO1305)hsp17therm-ORF-fw TTGAATTCCATTATTGCCGGGGCCGTC
(*pBO1347)hsp17therm-ORF-rv TTCCGTGGCGGTCCGTAGGGAC
(*pBO1347)rep-toeprinting-fw CACATCAGGAGTTGATTATGTCTCTCATTC
(*pBO1349)rep-toeprinting-rv GAATGAGAGACATAATCAACTCCTGATGTG
(*pBO1349)derep-toeprinting-fw CAAGGGTAATCAATTGGTTCCACACATCAGG
(*pBO1348)derep-toeprinting-rv CCTGATGTGTGGAACCAATTGATTACCCTTG
(*pBO1348)fw-hsp17-probe TGTCTCTCATTCTTTACAAT (Northern blot probe)
rv-hsp17-probe TAATACGACTCACTATCATTTATTAGGAAAGCTGAAC (Northern blot probe)
fw-bgaB-probe AGAGCAATGGCCAGAGGAAA (Northern blot probe)
rv-bgaB-probe TAATACGACTCACTATAGATCGGCAAAGAATCTGGAT (Northern blot probe)
up-fw GCGGCTAGAAATGTAATTTCGGCAATC (segregation analysis via PCR)
cr-rv GTTAGGATACCGGCATCGTAATTAGC (segregation analysis via PCR)
cr-fw GCTAATTACGATGCCGGTATCCTAAC (segregation analysis via PCR)
down-rv GACAACTTTTTCAGCAGTCCATTCCCATGG (segregation analysis via PCR)
a Introduced restriction sites are underlined. * Corresponding plasmids
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Table SII. Absolute levels of bgaB fusion constructs
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E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader is based on nucleotide slippage and pseudoknot
formation
Jens Kortmann, Gregor Weber, Karl E. Klose
and Franz Narberhaus
in preparation
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
15
Temperature-mediated translational regulation by the
Vibrio cholerae toxT 5’ mRNA leader is based on nucleotide
slippage and pseudoknot formation
Jens Kortmann1, Gregor Weber†, Karl E. Klose† and Franz Narberhaus1
1Lehrstuhl für Biologie der Mikroorganismen, Ruhr-Universität Bochum, 44780 Bochum,
Germany
†South Texas Center for Emerging Infectious Diseases and Department of Biology, The
University of Texas at San Antonio, San Antonio, Texas
Running title: Vibrio RNA thermosensor
Keywords: riboregulator; FourU RNA thermometer; post-transcriptional control; pseudoknot;
virulence; ToxT; autoregulatory loop
1Corresponding author: Franz Narberhaus, Lehrstuhl für Biologie der Mikroorganismen,
Ruhr-Universität Bochum, Universitätsstrasse 150, NDEF 06/783, 44780 Bochum, Germany.
Tel: +49 (0)234 322 3100; Fax: +49 (0)234 321 4620; E-mail: franz.narberhaus@rub.de
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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Thermo-sensitive, cis-regulatory 5‘ untranslated regions (UTRs) of mRNAs modulate
virulence gene expression in pathogenic microbes such as Listeria monocytogenes or
Yersinia pestis by controlled melting of the Shine Dalgarno sequence. Here, we
present a novel regulatory mechanism in a Vibrio cholerae RNA thermosensor. The 68-
nucleotide FourU element resides upstream toxT coding for a virulence master
regulator and, in contrast to all other virulence thermometers, inhibits ribosome
binding and associated translation at 37°C when tested in vitro and in E. coli. Proper
function of the toxT leader is based on two regulatory elements, the FourU
thermometer and a flexible top portion module. We pinpoint the nucleotides that
contribute to sequestration of the ribosome binding site at 37°C. Nucleotide slippage
and subsequent pseudoknot formation between two palindromic sequences result in
formation of an untranslatable tertiary structure. The repressive RNA-inherent feature
of the toxT 5‘-UTR is overcome by an unidentified mechanism in V. cholerae strains.
As Vibrio virulence is ruled by a multifaceted regulatory network, we propose that a
Vibrio-specific factor blocks the conformational switch when expression of toxT and
the dependent virulence factors is needed. Our findings suggest that the toxT
thermometer contributes to Vibrio pathogenicity by modulating virulence gene
expression at different stages of host infection.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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Introduction
The Gram-negative aquatic microbe Vibrio cholerae is the etiological agent of cholera, an
epidemic plague that afflicts several million people per year (1). Successful colonization of
the human intestine is crucially driven by the two primary virulence factors cholera toxin (CT)
and the toxin-coregulated pilus (TCP), whose variable expression is under control of a tightly
regulated transcriptional cascade known as the ToxR regulon (2,3). This virulence cascade
culminates in expression of ToxT, an AraC-family regulator, which directly activates
transcription of CT and TCP genes by binding to degenerate 13 bp sequences called
toxboxes (4-8). ToxT additionally activates transcription of two chemoreceptor genes, tcpI
and acfB, located within the Vibrio Pathogenicity Island, which contribute to intestinal
colonization (9). Thus, ToxT is an inevitable prerequisite and master regulator of Vibrio
virulence.
Multiple environmental stimuli feed into the Vibrio virulence regulon, thereby
modulating activity, (promoter)-specificity and abundance of the ToxT master regulator
(10,11). Sensitivity of ToxT to temperature, intestinal bicarbonate and bile (10,12-14) reflects
situations that occur either in aquatic reservoirs (<30°C), or distinct niches in the human body
(37°C, bile, bicarbonate). Bicarbonate might act as a positive effector molecule directly
modulating the ToxT protein (12). Recently solved crystal structures revealed that
monosaturated fatty acid compounds in human bile govern dimerization of the ToxT protein
and thereby its promoter specificity (14). A temperature upshift from 30 to 37°C down-
regulates ToxT-dependent transcription of the CT encoding ctxA gene (13-fold) (10). The
exact mechanism by which temperature affects synthesis or performance of the ToxT protein
itself remains unclear. ToxR and TcpP, two inner membrane proteins, initially activate toxT
transcription (15-17). Both require accessory proteins, ToxS and TcpH respectively, for
maximal activity (16). This primes an autoregulatory loop in which ToxT positively influences
its own transcription via the tcp promoter (18) (Fig.1). It is not clear how the autoregulatory
loop is shut off. Indeed, much research has been focused on the dissection of transcriptional
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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and post-translational events responsible for toxT gene expression, e.g. recent antimicrobial
drugs target either the ToxT protein (19), or act on DNA level by suppressing toxT
transcription (20).
During the last decade, the universality of post-transcriptional regulation by means of
regulatory RNAs became evident (21-26). In V. cholerae, small RNAs are widely used as key
regulatory elements responsible for the regulation of cellular processes such as
pathogenicity (27-29) or quorum sensing (30-32), mostly by decreasing the abundance of
target mRNAs. Exploiting Vibrio datasets obtained by deep-sequencing-based
transcriptomics gave rise to the discovery of multiple sRNAs and cis-regulatory 5‘-leaders
(33,34). Described Vibrio riboswitches undergo conformational changes upon binding of a
small molecule ligand to their aptamer domain (35-38). This affects formation of transcription
terminators or alters accessibility of the ribosome binding site (RBS) (39,40).
RNA thermometers are translational control devices that reside in the 5‘ untranslated
region (UTR) of bacterial heat shock or virulence genes and exclusively respond to
temperature alterations (41). At low temperatures the RBS-inherent Shine Dalgarno (SD)
sequence is blocked by base pairing. An upshift to human body temperature (37°C, virulence
genes) or higher (42°C, heat shock genes) usually liberates the SD sequence and facilitates
formation of the ternary translation initiation complex. RNA thermometer functionality within a
physiological temperature range is achieved by a delicate composition of its secondary
structure comprised of canonical and non-canonical base pairs, loops and bulges (42-46).
Figure 1. The ToxT master regulator is centrally positioned within Vibrio virulence networks. Transcription of toxt mRNA is activated by ToxR/S and TcpP/H. The ToxT protein activates
transcription of CT and TCP encoding genes (ctx and tcp, respectively). ToxT is also capable to activate its own transcription via the tcpA promoter (autoregulatory loop). This work focuses on
dissecting temperature-mediated regulation of toxT mRNA.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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In this work, we characterize the first virulence thermometer crucially dependent on
tertiary structure rearrangements. An RNA thermosensor-like structure is built-into the 5‘-
leader of the Vibrio toxT messenger. While sequestration and liberation of the SD sequence
is based on pairing with four uridines as in typical FourU thermometers (46), this element
favours translation at low (30°C) but not at elevated temperature (37°C) when tested in vitro
and in E. coli. We demonstrate by in vitro structure probing and toeprinting experiments that
repression of translation initiation at 37°C is based on nucleotide slippage and pseudoknot
formation. Strikingly, negative regulation at host temperature is overcome when tested in the
background of highly pathogenic Vibrio strains.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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Materials and Methods
Strains and growth conditions
E. coli (DH5α) and V. cholerae cells (Classical biotype strain KKV598 and El Tor biotype
strain C6706) were grown at 30 or 37°C in Luria-Bertani (LB) medium supplemented with
ampicillin (ApE.coli, 150 µg/ml; ApV.cholerae, 100 µg/ml). For induction of the pBAD promoter in
strains carrying translational lacZ fusions, 0.01% (w/v) L-arabinose was added.
Plasmid construction
The V. cholerae toxT 5‘-UTR was amplified via PCR (for primer sequences, see Table 1),
using chromosomal DNA from Classical N16961 strain and cloned into pUC18 (pBO1294).
The toxT-lacZ fusion (pBO1296) was constructed by transfer of the toxT 5’-UTR from
pBO1294 upon NheI/EcoRI digestion into the corresponding site in pBAD18-lacZ481 (Table
1). Site directed mutagenesis to generate pBO1826 (U5C), 1827 (∆G28), 1828 (∆14-48),
1829 (U7C), 1830 (SCE4), 1831 (G28C), 1839 (Kolkata), 1840 (SCE200), 1841 (C55G),
1842 (GG28/28CC) and 1843 (∆GG28/29) was performed according to the instruction
manual of the QuikChange® mutagenesis kit (Stratagene, La Jolla, USA). Plasmid pBO1294
served as a template for PCR with mutagenic primers (Table 1). The inserts containing
mutated toxT UTRs were isolated upon NheI/EcoRI digestion and cloned into the
corresponding site upstream of the lacZ gene. The correct nucleotide sequence was
confirmed by automated sequencing (eurofins, Martinsried, Germany). Chromosomal
N16961 DNA was provided by Prof. Dr. Joachim Reidl (University of Graz, Austria).
RNA detection
Preparation of total RNA from E. coli and Northern analysis followed previously published
protocols (47). RNAs were detected with a DIG-labeled lacZ RNA probe (DIG-HIGH prime
labeling kit; Roche Applied Science, Mannheim, Germany; see Table 1). Signals were
detected with a ChemiImager™ Ready (Alpha Innotech, Biozym, Wien, Austria).
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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In vitro transcription
RNAs were synthesized in vitro by runoff transcription with T7 RNA polymerase from
linearized plasmid templates. Plasmids pBO1299, 1308 and 1309 (linearized with MlsI) were
used to generate RNA for enzymatic structure probing and plasmids pBO1300, 1307 and
1331 (linearized with Bsp681) to generate RNA for toeprinting experiments (Table 1).
Structure probing experiments
RNA structure formation alongside a temperature gradient (20-50°C) or at either 28 and 42°C
was compared. RNA was 5’-end labeled as described (48). Partial digestion of 5’-end-labeled
RNAs with ribonucleases T1 (0.004U), V (0.008U) was conducted as described (this study).
For chemical probing, labeled RNA was mixed with 2 µl 5x lead buffer (250 mM tris acetate
pH 7.5, 25 mM Mg acetate; 250 mM Na acetate) and 1 µg tRNA in a total volume of 10 µl.
Lead(II) probing assay was conducted as described (this study). RNA fragments were
separated on denaturing polyacrylamide gels (8 or 15%). Alkaline ladders were generated as
described previously (48). The Aida Image Analyzer software (version 4.19) was used for
quantification of RNase cleavage intensity (Fig. 6).
Toeprinting analysis
Primer extension inhibition experiments were carried out using 30S ribosomal subunits,
target mRNA and tRNAfMet basically according to Hartz et al. (49). The 5’-[32P]-labeled toxT-
specific oligonucleotide toxT-runoff-toeprinting (Table S1) was used as a primer for cDNA
synthesis. An aliquot of 0.08 pmol mRNA (5’-UTR plus 60 nt of the toxT coding region)
annealed to the oligonucleotide was incubated for 10 min at 30 or 37°C, respectively,
together with 16 pmol of uncharged tRNAfMet (Sigma-Aldrich, St. Louis, Missouri). 6 pmol of
30S subunits or ribosome-buffer (TICO buffer, negative control) was added and incubation
for another 10 min followed before 2µl of MMLV-Mix (VD+Mg2+-buffer, BSA, dNTPs and
MMLV reverse transcriptase (USB, Cleveland, Ohio)) was added. cDNA synthesis was
performed at 30°C. Reactions were stopped after 10 min by adding formamide loading dye
and aliquots were separated on a denaturing 8% polyacrylamide gel.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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β-Galactosidase assays
β-Galactosidase activities of E. coli and V. cholerae strains carrying lacZ-fusions (pBO1296,
1826, 1827, 1828, 1829, 1830, 1831, 1839, 1840, 1841, 1842 and 1843) were measured as
described previously (50). The cells were shifted from 30°C to prewarmed flasks at 37°C for
various time lengths as indicated in the figure legends.
In silico RNA structure prediction
RNA secondary structures were predicted by using the mfold server (http://
frontend.bioinfo.rpi.edu/applications/mfold/cgi-bin/rna-form1.cgi) running version 3.2 (51) and
the RNAsubopt package (http://mobyle.pasteur.fr/cgi-bin/portal.py?form=rnasubopt; (52). For
pseudoknot predictions we used the pnotsRG program from the Bielefeld University
Bioinformatics Server (http://bibiserv.techfak.uni-bielefeld.de/pknotsrg/; (53).
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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Results
Functional analysis of the Vibrio toxT 5‘ element
The entire toxT 5‘-UTR (as found in Classical and El Tor strains) including its determined
ToxR-dependent transcription start site (54) and the AUG triplet was used for an in silico
folding approach (51). Consisting of 68 nucleotides with a calculated overall free energy of
-12.0 kcal mol-1 (51), the estimated toxT 5‘ leader structure comprises a stable stem
harbouring the SD (AGAG) and anti-SD FourU-motif, the internal asymetric loop L1 and the
top loop L2 (Fig. 2A). The AUG is separated from the basis of the stem by a palindromic five-
nucleotide stretch.
The Escherichia coli reporter plasmid system pBAD18-lacZ481 (55) was used to
examine temperature-dependent regulation of translation by the toxT 5‘-UTR. The leader
sequence was cloned between the L-arabinose-inducible pBAD promoter and the lacZ
reporter cassette (Fig. 2B). As Vibrio is found either in marine habitats or human hosts, we
checked for reporter gene expression at 30 and 37°C. To our surprise, the β-galactosidase
activity of 21 MU at 30°C was drastically reduced to merely 2 MU by a sudden shift to 37°C
(Fig. 2C). This shut-off of reporter gene activity by the toxT 5‘-UTR at human body-
temperature was contrasted by the control experiments with the Salmonella agsA FourU
thermometer (46) and its >5-fold induction upon temperature upshift. Even a translational
fusion to the 5‘-UTR of the E. coli housekeeping gene gyrA (coding for the DNA gyrase)
showed 2-fold induction at 37°C. Subsequent Northern analysis demonstrated that the
decline in reporter gene activity at 37°C was due to translational control by the toxT leader
rather than to altered transcript levels (Fig. 2D). This indicates tight and efficient regulation by
the Vibrio toxT 5‘-leader at the post-transcriptional level.
The top portion of toxT leader mRNA undergoes temperature-mediated slippage, enabling
pseudoknot formation in close vicinity to SD sequence and AUG start site
As reduction of translation initiation at elevated temperatures is a rare feature of cis-
regulatory mRNAs, we addressed the structural changes of the toxT 5‘-UTR in detail. The
toxT 5‘-UTR was synthesized by T7 RNA polymerase-mediated in vitro transcription and its
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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Figure 2. Translational control by the toxT 5‘-UTR in E. coli. (A) Predicted secondary structure (Zuker, 2003) of the entire toxT leader. The start codon (AUG, bold letters) is located 69 nucleotides
downstream of the transcription start site. The SD and 4U (anti-SD) sequences, loop 1 (L1) and loop 2 (L2) are labeled. (B) Schematic representation of the reporter gene fusion on plasmid pBAD18-
lacZ481. Additional nucleotides inserted due to the position of the NheI cloning site relative to the pBAD promoter are indicated by a box upstream of the 5’-UTR. The artifical nucleotides at the 5’ end
of the toxT transcript do not influence RNA folding and expression of the gene (data not shown). (C) Expression of translational lacZ reporter fusions (Miller Units, MU) to the toxT 5‘-UTR. E. coli DH5α
cells containing plasmid pBO1296 carrying the toxT-lacZ fusion were grown in LB medium at 30°C and either kept at this temperature (white columns) or transferred to 37°C (black columns) for 30 min
before β-galactosidase activity was measured. A Salmonella agsA-lacZ fusion (FourU thermometer; Waldminghaus et al. 2010) was used as a positive control, while E. coli gyrA-lacZ served as a
negative control. All experiments were repeated at least in triplicate. (D) mRNA levels of toxT, agsA and gyrA-lacZ fusions at 30 and 37°C. Total RNA was extracted from E. coli cultures incubated at
either 30 or 37°C. Equal amounts were separated on a 1.2% denaturing agarose gel and Northern blot experiments were carried out using digoxygenin-labeled RNA probes to detect lacZ transcripts.
Ethidium-bromide stained 16S rRNAs from the gel before blotting are shown as loading control. The lacZ fusion transcripts run at 3 kb.
A B
C
D
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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structure was mapped by enzymatic and chemical probing at a temperature gradient from 20
to 50°C. The cleavage pattern after limited digestion with different RNases indicated that the
in silico predicted structure (Fig. 2A) only applies to low temperature conditions (20-30°C) but
undergoes subtle conformational changes in its top portion above 30°C (Fig. 3A). As
predicted, the two guanine residues G28 and G29 in loop L2 were sensitive towards RNase
T1, which cuts 3‘ of single-stranded guanines, from 20 to 30°C. However, above 30°C both
nucleotides became increasingly susceptible to RNase V which is specific for double-
stranded (paired) regions (56). The RNAsubopt package (52) predicted suboptimal folding of
the toxT RNA at various temperatures (supplementary Fig. S1), providing additional in silico
evidence that G28 and G29 move into the top portion stem via a slippage-like mechanism as
in the synthetic theophylline riboswitch (57). The translational shut-down at 37°C by the toxT-
leader does not depend on sequestration of the SD sequence, as we detected T1 cleavage
of SD guanines from 30°C upwards. Poor sensitivity of nucleotides G57 and G59 at 20 and
25°C suggested pairing with the anti-SD region at low temperatures as in other FourU
thermometers. Another structural conversion above 30°C occurred in close proximity to the
SD. The palindromic stretch ACGCA motif (PS1) became increasingly sensitive towards
RNase V above 30°C. Lead probing supported the enzymatic probing results. Lead is a
single-strand-specific probe that induces cleavage of the sugar-phosphate backbone. The
PS1 nucleobases appeared to be single stranded at 20, 25 and 30°C but were protected
against cleavage at higher temperatures (Fig. 3B). Sequestration of the PS1 side was
accompanied by base pair slippage in stem loop L2 beyond 30°C as nucleotides C32, U33
and C34 were progressively attacked by lead ions. This slippage liberated a second
palindromic sequence, UGCGU (PS2), with perfect complementarity to PS1 (Fig. 3C).
Potential pseudoknot (PK) interaction between the upstream residing PS1 sequence and the
temperature-sensitive PS2 region at 37°C is supported by in silico analysis using the
pknotsrg program. The resulting tertiary structure might prevent docking of the 30S ribosome
to the SD sequence, thereby shutting off translation initiation at 37°C.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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A B
C
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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Figure 3. Enzymatic and chemical „melting curve“ probing of the toxT leader. (A-B) Enzymatic hydrolysis and lead(II)-induced cleavages performed on 5‘-end-labeled toxT 5‘-UTR alongside a
temperature gradient (20-50°C, using a 5°C-stepped gradation). The conditions for RNase and lead(II) concentrations were as follows: (A) RNase T1 (0.004U), RNase V (0.008U); (B) lead(II) (10
mM). RNA fragments were separated on 8% (A) or 8 and 15% (B) polyacrylamide gels. Lanes N, controls without RNase or modifying agents; lanes 50, RNase cleavages under denaturing conditions;
lanes L, alkaline ladder. SD (Shine Dalgarno), L1 (loop 1), PS1/2 (Palindromic Sequence 1/2), top portion and anti-SD regions are indicated; PK (Pseudoknot). (C) Computer predicted (Wuchty et al.,
1999; Zuker, 2003) secondary structures of the toxT 5‘-UTR at 30 (blue) and 37°C (red). Direction of nucleobase slippage is depicted by arrows. PK formation between PS2 and PS1 at 37°C is visualized
by dotted lines. Nucleotides of interest are encircled. The AUG start codon is marked by bold letters.
The toxT 5‘-UTR inhibits ribosome binding at 37°C
To examine temperature-controlled ribosome binding to the toxT 5‘-leader, we performed
toeprinting (primer extension inhibition) assays at 30 and 37°C. Ternary complex formation
between initiator tRNAfMet, 30S ribosomes and the toxT transcript (68 nucleotides 5‘-UTR +
60 nucleotides coding region) was defined by extension of a radio-labeled primer.
Prematurely terminated cDNA products (toeprints) corresponding to the +14 position
downstream of the AUG triplet were expected if ribosome was bound to the toxT RNA.
Consistent with the reporter gene fusion results (Fig. 2C), ribosome binding evidenced by a
prominent toeprint associated with reduced yield of full-length cDNA at 30 but not 37°C (Fig.
4). A structural rearrangement at 37°C is supported by the appearance of two additional
Figure 4. Temperature-dependent ribosome binding to the toxT leader. Toeprinting was carried out on 2 pmol RNA as described in Materials and Methods. The absence (-) or presence (+) of 30S
subunits is indicated. Terminated reverse transcription products (toeprints) at position +14 relative to the A of the start codon and full-length products are pointed out by arrows. TGCA refers to a
sequencing ladder generated with the same oligonucleotide as in the toeprint experiments. The position of the start codon is boxed. Position of the SD (Shine Dalgarno) sequence is indicated.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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toeprint signals at positions of guanine 38 and cytosine 32 in the presence and absence of
30S ribosomes.
Characterization of rationally designed and natural oriented toxT leader variants
Inhibition of toxT translation initiation is based on secondary and tertiary structure
rearrangements interfering with ribosome access. Since the in vitro experiments suggested
that this happened in an RNA-only fashion, point mutations in critical RNA regions should
have an impact on toxT thermosensing. Selected sites for oligonucleotide-directed
mutagenesis are outlined in Fig. 5A, including those of naturally occurring toxT variants,
which deviate from Classical/El Tor leader sequences. The alignment includes 5‘-UTRs from
less infectious environmental strains and recent patient isolates (Kolkata strains, (Chatterjee
et al., 2009) (Fig. 5B). All four Kolkata variants SC132, SC124, SC107 and SC72 carry
changes (G3A, U5C and U13C) in or around the anti-SD sequence as does the
environmental strain SCE200 (G3A and U13C). SCE4 contains a substitution in the top
portion loop L2 (C32U), one downstream of the SD sequence (A61G) and a deletion of
bulged cytosine 55 (ΔC55).
The first set of rational mutations (M1-M4) directly targeted the two purines G28 and
G29 to override translational repression by blocking temperature-induced base pair slippage
of loop L2. Indeed, M1 (ΔG28/G29), M2 (GG28/29CC), M3 (ΔG28) and M4 (G28C)
prevented repression at 37°C suggesting impaired liberation of the PK forming PS2 site (Fig.
5C). M1 to M4 exhibited increased β -galactosidase activity upon a shift from 30 to 37°C.
Neither of the four variants was transformed into a typical RNA thermometer, like the
Salmonella agsA 5‘-UTR, which revealed a 5.9-fold induction. Interestingly, efficient heat
induction was achieved by removal of the entire stemloop II through deletion mutation M5
(Δ14-48).
With a 6.5-fold induction from 19.8 MU (30°C) and 124.9 MU (37°C) M5 acted as bona fide
RNA thermometer (for secondary structure prediction see supplementary Fig. S2). This
indicates that the toxT 5‘-UTR is a bipartite thermosensor, in which stemloop II prevents the
otherwise favourable melting of stemloop I at 37°C. To study the contribution of the anti-SD
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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sequence we substituted substituted G-U base pairs by more stable G-C base pairs. M6
(U5C) and M7 (U7C) both exhibited tighter repression at both temperatures. Exchange of the
bulged cytosine immediately upstream of the SD sequence (C55) to a G (M8) had little effect
on β -galactosidase activity at 30 and 37°C suggesting that this residue plays no significant
role in the toxT 5‘-UTR.
The natural toxT 5‘-UTR variants revealed different temperature responses. While the
M9 (Kolkata) and M11 (SCE200) variants still repressed reporter translation (although less
Figure 5. Characterization of rationally designed and natural oriented toxT leader variants. (A) Site-directed mutations M1 to M11 and the exchanged nucleotides are denoted in red letters. (B) Alignment of toxT 5‘-UTR variants from isolated environmental and clinical strains (Kolkata) to the El Tor O395 sequence (which we used as WT toxT leader sequence in this work). Deviating nucleotides
are marked with bold letters. The respective position is underlined in the WT sequence. (C) Expression of the translational lacZ reporter fusions (Miller Units, MU) to various toxT 5‘-UTRs.
Experiments were conducted as described (legend Fig. 2; Materials and Methods). The bars indicate the means of at least three independent experiments, and the error bars indicate standard deviations.
Induction / repression rates are shown above each fusion. (D) In vitro transcribed toxT thermometer variants M1 and M2 were used for toeprinting analysis at 30 and 37°C as described (legend Fig. 4;
Materials and Methods). The arrows indicate up- or down regulation.
A B
C
D
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effective than the WT), M10 (SCE4) lacked the ability to repress translation at 37°C. This
might hint for differences in virulence gene regulation in evolutionary diverse Vibrio strains.
To obtain further evidence for the requirement of G28 and G29 in translational
repression at 37°C, we conducted toeprinting analysis of M1 and M2 (Fig. 5D). Both variants
allowed equally effective docking of the ribosome to the SD sequence at 30 and 37°C.
Deletion of G28/G29 (M1) or substitution by cytosine residues (M2) prevented formation of
ribosome-blocking structures in good agreement with the abolished reporter gene induction
in the lacZ assay. To validate the effect of G28 and G29 on base pair slippage, we compared
the structure of the M2 RNA with the WT transcript (Fig. 6). The M2 RNA revealed
comparatively increased rigidity of stemloop L2, visible by enhanced RNase V cleavage at
positions C34, U35, C37, G38 and U39. Thus, a proper base pair slippage is presumably not
longer possible. The SD residing nucleotides G57 and G59 were attacked by RNase T1 to a
greater extent, when compared to the WT RNA. This suggested an enhanced susceptibility
of the RBS site, due to failed PK formation in the M2 variant.
Figure 6. Comparative structure probing analysis of toxT WT and M1 5‘-UTR. Enzymatic cleavages performed on 5‘-end-labeled toxT 5‘-UTR at 30 or 37°C. The conditions for RNase T1 and
RNase V were as described (legend Fig. 3; Materials and Methods). Bars mark the fold difference in RNase V (black) or T1 (grey) cleavage intensity at 37°C of M2 variant relative to the WT. Positions of
top portion, SD (Shine Dalgarno) and start (AUG) codon were indicated relative to the transcription start site.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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The Vibrio case: high induction by the toxT 5‘-leader at host body temperature (37°C)
Spatio-temporal regulation of virulence gene expression is inevitable to avoid early immune
responses and start host invasion at the right time point (2,58). Somewhat counterintuitively,
the toxT 5‘-UTR successfully repressed formation of a translation initiation complex at 37°C
in vitro and in E. coli by an RNA-only mechanism. However, when checked in the
background of a Classical V. cholerae strain the toxT cis-element enabled a 19-fold induction
of the lacZ reporter gene upon a shift from 30 to 37°C (Fig. 7). The Vibrio El Tor strain
background caused higher β-galactosidase activity at low temperature resulting in a 3.2-fold
induction.
The conversion of a repressing cis-element into an activating element suggests that the
RNA-inherent capacity of the toxT 5‘-UTR to repress translation at 37°C is overcome by a yet
undefined, trans-active factor in V. cholerae.
Figure 7. Translational control by the toxT 5‘-UTR in Vibrio. V. cholerae KKV598 (Classical Ogawa strain) or C6706 (El Tor Inaba strain) cells containing plasmid pBO1296 were grown in LB
medium at 30°C and either kept at this temperature (white columns) or transferred to 37°C (black columns) for 30 min before β-galactosidase activity was measured. Induction rates are indicated. All
experiments were repeated at least in triplicate. Arrows indicate upregulation.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
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Discussion
Hallmarks of the toxT thermosensor
Until recently, virulence gene expression in V. cholerae was thought to be controlled mainly
at the level of transcription initiation by activating or repressing proteins (2). Although several
regulatory RNAs have been described in Vibrio, most of them are related to physiological
processes (30,32,59,60). Song et al. recently described a regulative RNA with probable
impact on Vibrio pathogenesis. A mutant strain lacking the small RNA vrrA exhibited
increased expression of the ToxT-dependent virulence factor TCP (28). The trans-activity of
vrrA was shown to depend on the RNA chaperone Hfq (29).
This study describes the discovery of a cis-regulatory RNA found upstream of the
virulence master regulator ToxT in highly pathogenic V. cholerae strains. The toxT leader is
the first RNA thermometer found in a Vibrio species. The isolated element represses
translation initiation at 37°C in vitro and in E. coli by sequestration of the RBS at 37°C in an
RNA-only mechanism. The structure-based negative regulation at human body temperature
was overcome in the two most-pathogenic Vibrio biotypes, Classical (strain KKV598) and El
Tor (strain C6706). We propose that a trans-acting Vibrio-factor is involved in this process
(see below).
Having a length of 68 nucleotides, an internal loop and a paired RBS, the toxT 5‘-UTR
comprises all features of a functional RNA thermosensor (61,62). Since four uridine residues
pair with the SD sequence, it belongs to the class of FourU thermometer elements like
Salmonella agsA (46) or Yersinia lcrF (63). In contrast to agsA or lcrF, the toxT 5‘-UTR is
organized in a bipartite fashion. Typical thermometer-like melting and translation initiation at
elevated temperatures is possible with hairpin I alone. Hairpin II, however, prevents
translation at 37°C in E. coli and in vitro by a complex mechanism involving nucleobase
slippage and pseudoknot formation. It has been reported that single nucleotide slippage in a
rationally designed theophylline riboswitch can be sufficient to prevent translation initiation
(57). This was achieved by coupling the theophylline aptamer domain to a synthetic induction
module developed by the Breaker lab (64) and clearly demonstrates that subtle
conformational changes in an RNA element may alter functionality in vivo (57,65). Here, we
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
18
demonstrate slippage of a complete nucleotide chain. At elevated temperatures, sliding of
nucleotides in the toxT top portion element liberates a palindromic stretch of five nucleotides
(UGCGU, PS2) with perfect complementarity to another palindromic sequence (ACGCA,
PS1) between the SD sequence and the AUG start codon. Structural reorganization of the
top portion element dictates the accessibility of the adjoining FourU thermometer module by
PK formation between PS2 and PS1 (Fig. 3). The PK was stable enough to prevent binding
of 30S ribosomal subunits at 37°C in toeprinting experiments (Fig. 4). The fact that deletion
of the stem II module turns the repressing toxT element into a classical FourU thermometer
might be a hint that the toxT thermosensor evolved from a heat-sensing RNA thermometer,
to which an additional control module has been added during co-evolution with the human
host.
Functionality of both the slippage module and the toxT FourU thermometer was
sensitive towards single point mutations. Substitution of two guanine residues in the L2 loop
impaired nucleotide chain slippage and interfered with formation of RBS masking structures.
The mutants were able to bind 30S ribosomal subunits at 37°C, which was also evident by
enhanced accessibility of the SD sequence towards RNase T1 and elevated reporter gene
activity. In summary, the toxT 5‘-UTR is an unique RNA element consisting of a slippage
module and the FourU thermometer and a downstream box whose concerted action controls
translation of the virulence master regulator ToxT.
The first six cholera pandemics (1817-1923) were caused by the Classical, and the
ongoing seventh pandemic (1961-present) by the El Tor biotype (66).
The toxT 5‘-UTR is identical in both biotypes. Interestingly, there are differences in the 5‘
leader sequences of the less infectious Vibrio strains SC132, SC124, SC107 and SC72 that
were recently isolated from hospitalized diarrheal patients in Kolkata, India (67). These
strains lack ~6% of the V. cholerae genome and have different virulence factors including
alleles of the toxT gene. The 5‘-UTR of alternate toxT genes of all four Kolkata strains
deviate in three nucleotides from the WT allele. The Kolkata consensus 5‘-UTR still
repressed E. coli reporter gene expression but less efficient than the toxT WT thermometer
(Fig. 5). We obtained equal results for an environmental toxT leader variant (SC200), only
the SC4 strain completely lacked negative regulation. How these different toxT thermometer
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
19
variants influence toxT expression in Vibrio and how this affect virulence remains to be
studied.
Temperature-controlled toxT expression in V. cholerae
ToxT is centrally positioned in the V. cholerae virulence cascade. Since a toxT deletion
mutant highly attenuated for virulence (68), every alteration in abundance of the ToxT will
have an impact on virulence gene expression. ToxT transduces environmental signals
detected by a complex sensory system including ToxR/ToxS, TcpP/TcpH and the outer
membrane proteins OmU/OmpT (2,16). Bile and temperature directly affect expression of
ToxT-dependent genes (10) as well as other intestinal compounds (12,14). On basis of our
study, we postulate another layer of control exerted by the toxT thermosensor. There is basal
ToxR-dependent expression of toxT under environmental conditions (69). Seasonal warming
of V. cholerae aquatic reservoirs to 30°C might be sufficient to melt the toxT SD sequence
(Fig. 3), thereby facilitating translation initiation and expression of the ToxT protein (Fig. 8a).
The present ToxT activator starts an autoregulatory loop (18) resulting in constant increase of
toxT mRNA at elevated environmental temperatures (30°C), priming Vibrio for host contact. It
is known that warming of the aquatic environment favours survival and growth of V. cholerae.
The importance of this ecological parameter is reflected by annual increases of cholera
incidents in Southeastern Africa (70). The multiplication and persistence of V. cholerae in the
aquatic environment is correlated with massive proliferation of phytoplankton at 30°C (71) on
which Vibrio species form biofilms which enhances the chance of ingestion by the human
host via contaminated water. Ingestion by the warm-blooded human host leads to a sudden
increase in temperature (37°C). This induces tertiary structure rearrangements in the toxT 5‘
leader. Concomitant PK formation shuts off expression of the ToxT protein and, inevitably,
downregulates all ToxT-dependent virulence factors (Fig. 8b) which might protect Vibrio from
the host innate immune defense, especially the human complement system (72,73). This
RNA-only repressive mechanisms can be counteracted by an unknown Vibrio-specific factor
(VSF) (Fig. 7). Binding of the VSF might hinder nucleobase slippage and PK formation
granting the ribosome access to the SD site. Switching on toxT expression induces the
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
20
virulence factors TCP and CT and, by this, initiates the final phase of host-invasion (Fig. 8c).
Most likely, VSF expression and docking to the toxT slippage module happens before the
intestinal entry, because TCP is needed for tethering of Vibrio cells to prevent sheering
forces (2). The toxT expression is shut down at low temperatures of cold marine habitats
(<20°C), simply because the base pairing between the FourU and the SD sequence is too
stable (Fig. 3) to allow binding of the ribosome (Fig. 8d).
The nature of the VSF is unknown. It could either be a protein or a regulatory RNA.
An example of a complex virulence thermometer is the L. monocytogenes prfA element,
which was initially reported to act by the classical melting mechanism (43). However, recently
the involvement of a S-adenosylmethionine (SAM) riboswitch, SreA, which represses
expression of prfA by binding to the thermometer region, has been reported (74). Screening
large-scale sequence-defined mutant libraries could help identifying the unknown VSF (75).
Figure 8. The toxT thermometer governs spatiotemporal expression of the ToxT virulence master regulator by temperature-mediated conformational changes. For detailed information,
see discussion.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
21
Given the involvement of a regulatory RNA, it might be included in previously reported data
sets (33,34).
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
22
Funding
This work was funded by a grant from the German Research Foundation (DFG priority
program 1258) to FN and by a fellowship from the Studienstiftung des Deutschen Volkes to
JK.
Acknowledgements
We thank Joachim Reidl (Graz) for providing V. cholerae chromosomal DNA. We are grateful
to Birgit Klinkert for preparation of 30S ribosomal subunits and to Ulla Aschke-Sonnenborn
for technical assistance. All members of the RNA group, Robert Giegerich (Bielefeld) and
Petra Dersch (Braunschweig) are acknowledged for critical comments on the manuscript and
helpful discussions.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
23
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Figure S1. Suboptimal folding of the toxT 5’-UTR. Dot-bracket annotations of the toxT leader RNA as emitted by RNAsubopt in silico prediction (Wuchty et al., 1999). The -11.54 free energy score
reflects the most stable structure (100%). Free energy is given in kcal mol-1.
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
29
Figure S2. Secondary structure prediction of toxT leader variant M5. In silico prediction of structure and overall free energy was realized with mfold (Zuker, 2003).
Table S1. Strains, plasmids and oligonucleotides used in this study
Strain, plasmid or oligonucleotide
Relevant characteristic(s) or sequence Source or reference
Strains
E. coli DH5α supE44 ΔlacU169 (Φ80lacZΔM15) hsdR17 recA1 gyrA96 Gibco-BRL
V. cholerae KKV598 Classical Ogawa strain, ΔlacZ Correa et al., 2000
V. cholerae C6706 El Tor Inaba strain, ΔlacZ, StrepR Thelin and Taylor, 1996
Plasmids
pUC18 Cloning vector, AmpR Norrander et al. 1983
pBAD18-lacZ481 Translational lacZ fusion vector, AmpR Waldminghaus et al. 2005
pBO1294 Vibrio toxT 5’-UTR (thermometer element) in pUC18 (QuikChange® template)
This study
pBO1296 Vibrio toxT 5’-UTR-lacZ fusion in pBAD18-lacZ481 This study
pBO1826 Vibrio toxT 5’-UTR-U5C-lacZ fusion in pBAD18-lacZ481 (M6) This study
pBO1827 Vibrio toxT 5’-UTR-ΔG28-lacZ fusion in pBAD18-lacZ481 (M3) This study
pBO1828 Vibrio toxT 5’-UTR-Δ14-48-lacZ fusion in pBAD18-lacZ481 (M5) This studypBO1829 Vibrio toxT 5’-UTR-U7C-lacZ fusion in pBAD18-lacZ481 (M7) This study
pBO1830 Vibrio SCE4 toxT 5’-UTR-lacZ fusion in pBAD18-lacZ481 (M10) This study
pBO1831 Vibrio toxT 5’-UTR-G28C-lacZ fusion in pBAD18-lacZ481 (M4) This study
pBO1839 Vibrio Kolkata toxT 5’-UTR-lacZ fusion in pBAD18-lacZ481 (M9) This study
pBO1840 Vibrio SCE200 toxT 5’-UTR-lacZ fusion in pBAD18-lacZ481 (M11) This study
pBO1841 Vibrio toxT 5’-UTR-C55G-lacZ fusion in pBAD18-lacZ481 (M8) This study
pBO1842 Vibrio toxT 5’-UTR-GG28/29CC-lacZ fusion in pBAD18-lacZ481 (M2)
This study
pBO1843 Vibrio toxT 5’-UTR-ΔGG28/29-lacZ fusion in pBAD18-lacZ481 (M1) This study
pBO1297 Salmonella agsA-lacZ fusion in pBAD-lacZ481 (Control C.1) This study
pBO1298 E. coli gyrA-lacZ fusion in pBAD-lacZ481 (Control C.2) This study
pBO1299 Vibrio toxT 5’-UTR-WT-in vitro transcripts for structure probing experiments, runoff vector (via MlsI site), pUC18-based
This study
pBO1308 Vibrio toxT 5’-UTR-M1-in vitro transcripts for structure probing experiments, runoff vector (via MlsI site), pUC18-based
This study
pBO1309 Vibrio toxT 5’-UTR-M2-in vitro transcripts for structure probing experiments, runoff vector (via MlsI site), pUC18-based
This study
pBO1300 Vibrio toxT 5’-UTR-WT-in vitro transcripts (+60 bp coding region) for toeprinting experiments, runoff vector (via Bsp681 site), pUC18-based
This study
pBO1307 Vibrio toxT 5’-UTR-M1-in vitro transcripts (+60 bp coding region) for toeprinting experiments, runoff vector (via Bsp681 site), pUC18-based
This study
pBO1331 Vibrio toxT 5’-UTR-M2-in vitro transcripts (+60 bp coding region) for toeprinting experiments, runoff vector (via Bsp681 site), pUC18-based
This study
Oligonucleotides
NheI-toxT TTTGCTAGCTCGATTTTAAGATAAC (*pBO1296)
toxT-EcoRI TTTGAATTCAGACATAATGTTAACTCC (*pBO1296)
toxT-M1-fw CTTTACGTGGATCTCTCTGCGTTTA (*pBO1843)
toxT-M1-rv TAAACGCAGAGAGATCCACGTAAAG (*pBO1843)
toxT-M2-fw CTTTACGTGGATCCCTCTCTGCGTTTA (*pBO1842)
toxT-M2-rv TAAACGCAGAGAGGGATCCACGTAAAG (*pBO1842)
toxT-M3-fw CTTTACGTGGATGCTCTCTGCGTTTA (*pBO1827)
toxT-M3-rv TAAACGCAGAGAGCATCCACGTAAAG (*pBO1827)
toxT-M4-fw CTTTACGTGGATCGCTCTCTGCGTTTA (*pBO1831)
toxT-M4-rv TAAACGCAGAGAGCGATCCACGTAAAG (*pBO1831)
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
30
toxT-M5-fw GATAACTTTATTTATTTATATATG (*pBO1828)
toxT-M5-rv CATATATAAATAAATAAAGTTATC (*pBO1828)
toxT-M6-fw TCGACTTTAAGATAACTTTACGTGG (*pBO1826)
toxT-M6-rv CCACGTAAAGTTATCTTAAAGTCGA (*pBO1826)
toxT-M7-fw TCGATTCTAAGATAACTTTACGTGG (*pBO1829)
toxT-M7-rv CCACGTAAAGTTATCTTAGAATCGA (*pBO1829)
toxT-M8-fw TTATATATCTTGAGAGTAGAACGCAATG (*pBO1841)
toxT-M8-rv CATTGCGTTCTACTCTCAAGATATATAA (*pBO1841)
toxT-M9-fw TCAACTTTAAGACAACTTTACGTGGATG (*pBO1839)
toxT-M9-rv CATCCACGTAAAGTTGTCTTAAAGTTGA (*pBO1839)
toxT-M10-fw ATGGCTTTCTGCGTTTATTTATATATCTGAGTGGAAC (*pBO1830)
toxT-M10-rv GTTCCACTCAGATATATAAATAAACGCAGAAAGCCAT (*pBO1830)
toxT-M11-fw TCAATTTTAAGACAACTTTACGTGGATG (*pBO1840)
toxT-M11-rv CATCCACGTAAAGTTGTCTTAAAATTGA (*pBO1840)
T7-toxT GAAATTAATACGACTCACTATAGGGTCGATTTTAAGATAACTTTACG (*pBO1299 and pBO1300)
toxT-runoff-SP TGGCCATTGCGTTCTACTCTGAAG (*pBO1299)
toxT-runoff-toeprinting
TCGCGAATATGTAAGTATCAAATTTACTC (*pBO1300)
fw-lacZ-probe CTGGCGTAATAGCGAAGAGG (Northern blot probe)
rv-lacZ-probe TAATACGACTCACTATTTGATGAAAGCTGGCTACAGG (Northern blot probe)
E. Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader
31
F. Discussion
1. Synthetic biology - molecular tailoring of biological systems at the nanoscale
A strategy in engineering design is the combination of refined parts that can perform a human-
defined function (53). Jacques Monod was one of the first scientists who proposed a circuit-like
connectivity of biological parts which collectively process logical operations (at the 1961 Cold
Spring Harbor Symposium). Only ten years later, the newly discovered restriction enzymes and
ligases were used to connect viral DNA with chunks of a microbial genome (54). With the new
millennium, breakthroughs in genomic research and genetic engineering have supplied the
technical inventory and methods necessary to physically construct and assemble biomolecular
compounds (55). Today, synthesis of 500 nucleotides DNA of choice costs no more than 150,-
Euros and is done within a week (information by MWG biotech AG, Martinsried, Germany).
Most synthetic biology efforts have focused on engineering regulatory networks that rely on
protein-DNA interactions to perform certain tasks (56). Recently, attention has shifted towards
applications of regulatory RNA molecules (57). In both eukaryotes and prokaryotes, ribonucleic
acids fulfill multifaceted functions due to their ability to adopt complex structures (1, 58-60).
2. RNA synthetic biology inspired by microbes: construction of translation initiators under
temperature control
Structural motifs in naturally occurring RNAs can be applied as new molecular parts for synthetic
biology to facilitate the development of novel tools and system devices that modulate cellular
functions (61). Often it takes a mix of different experimental procedures to obtain the desired RNA
module. Combined chemical genetics and genetic selection helped converting the adenine-sensing
Vibrio vulnificus add riboswitch into a synthetic device that allows tuneable, dose-dependent
control of gene expression in response to nonnatural synthetic small molecules (62). In this work,
we examined the possibilities in combining a bioinformatic approach with classical wetlab
experiments to design a functional, thermosensitive biobrick. In contrast to Dixon and colleagues,
we had no source material as we started. The naturally occurring λ cIII thermosensor, an RNA-only
system with an equilibrium between two alternative thermometer conformations (24, 63), inspired
us to generate a switch-like thermosensor. Our approach did not utilize any nucleotide sequences
or characteristic structural domains of λ cIII. We made use of certain RNA structure prediction
F. Discussion
16
programs (SWITCH by Flamm et al. (64); RNAfold and RNAheat of the Vienna RNA Package (65))
and error-prone mutagenesis (in vivo screening) to finally come up with a nucleotide sequence that
seemed to fulfill the fixed criteria: to fold into two alternative secondary structures upon
temperature alterations, the OFF (30°C, no translation) and the ON (42°C, translation) state.
However, structure probing experiments revealed that only one conformation of the synthetic
thermometer was present at both temperatures. This structure acts by melting like a typical
thermometer. To receive a simple genetic element, we had chosen a sequence length of only 45
nucleotides including the AUG codon. The regulatory principle of the λ cIII thermometer might not
be transferable to the rather small synthetic RNA device, as λ cIII required not only 85 nucleotides
5‘-leader sequence but also 45 nucleotides of the coding region to be fully functional. Furthermore,
the λ cIII messenger flipped into a SD-blocking conformation at high temperature (24), a feature
comparable to translational repression by the Vibrio toxT thermometer analyzed in this work, but
unwanted in our synthetic approach. Another RNA-based thermosensor that falls into two
alternative conformations and blocks translation at high temperature has been described in the E.
coli cspA gene (42). Here, the region involved also is rather long and reaches far into the coding
region of cspA (159 nucleotides UTR + 187 nucleotides coding region).
The small size of the synthetic thermometer might have contributed to the fact that it took
only two rounds of mutagenesis and in vivo selection to obtain a temperature-sensing RNA as
efficient as naturally occurring variants. This fits well to the accepted „early RNA-world“ hypothesis
(66), where simple RNA regulators were formed under prebiotic conditions by Darwinian evolution
(and primordal self-replication) (67-69). It is conceivable, that contemporary complex RNA
elements have evolved from a plethora of simple ancient RNA modules.
Such a „construction kit-like“ attempt has been used to generate a couple of synthetic RNA
thermosensors (70). In contrast to our approach, Neupert and co-workers desired a simple one-
stem thermosensor that gradually melts due to its weak stem-loop structure rather than a switch
between two mutually exclusive conformations. They used three key modules (1. SD sequence, 2.
anti-SD sequence and 3. a spacer/loop sequence between SD and anti-SD) that allowed a quick
replacement through unique restriction sites for de novo synthesis of a temperature-sensitive RNA
(71). Two main parameters were manipulated to adjust the melting temperature: stem stability
(strength of base-pairing in the stem) and the loop size. The synthetic U6 thermometer variant
performed „even better than the natural Listeria monocytogenes prfA thermometer“ in that
temperature inducibility was stronger and background expression was virtually absent (70). Like
prfA, the Synechocystis hsp17 5‘-leader (this work), Salmonella agsA (27), ROSE (35) and ROSE-
F. Discussion
17
like thermometers (29, 37) also exhibited a certain degree of translation initiation under non-stress
conditions even when the gene product was not urgently needed by the cell. This is probably due
to constant „breathing“ of the structure facilitated by special architectural features (see
introduction). Natural thermosensors might not be optimized for full repression because a certain
degree of „leakiness“ might be beneficial under physiological conditions. A basal level of translation
would allow immediate priming of autoregulatory loops, e.g. the prfA gene is regulated by PrfA
itself via an autoregulatory loop mediated by the upstream PrfA-dependent lacA promoter (72), a
principle also discovered in Vibrio cholerae (73) and discussed in context of the newly found toxT
RNA thermometer within this work.
Simplification and, above-all, decoupling of biological design principles are the key
elements of today´s synthetic biology. But, theoretical assumptions and information obtained in
vitro are not necessarily transferable to an in vivo situation. Implementation of a (decoupled)
synthetic RNA device into a non natural context harbours the (unpredictable) potential of unwanted
side effects or aberrant regulatory capacities. Recent work by the Cossart and Johansson groups
showed that the Listeria prfA thermometer is trans-regulated via a SAM-riboswitch element, SreA,
that represses translation by binding to the thermometer region (52). Therefore, it is conceivable
that an exchange of the prfA 5‘-UTR for another, synthetic or natural, thermometer would not
reconstitute a wild type phenotype. Foreign thermometers will presumably not fit into the Listeria
virulence regulon and therefore will not be recognized by the Listeria antisense-RNAs. Mis-
regulation of the Listeria virulence cascade might cause early activation of the human immune
response and attenuate or decrease infectivity.
Another example for the unpredictability of rationally designed RNA elements is a synthetic
riboswitch derived from the transcriptional Bacillus subtilis glycine riboswitch, which is a
transcriptional ON riboswitch (74). The synthetic variant contained an anti-SD sequence designed
to allow translational regulation (translational ON riboswitch) by base-pairing with the SD sequence
(75). Induction via glycine was still possible in gram-negative E. coli, but unforeseeable, the
synthetic RNA switch behaved like a translational OFF switch. Therefore, double-checking
functionality of synthetic thermometers under multiple conditions and in different genetic
backgrounds is recommended to finally receive a tight synthetic RNA biosensor, which
„communicates the state of a specific microenvironment within a sea of environmental conditions
(55)“. From this view, the short and simple Synechocystis hsp17 leader makes a perfect template
for synthetic RNA thermometer devices as we carefully examined its functionality under several in
vitro and in vivo situations (this work).
F. Discussion
18
3. Potential applications of the synthetic RNA thermometer module
Synthetic biology uses certain regulatory devices as composite parts to generate tools with
adjustable functionality. We envisage to employ synthetic thermometers as versatile modules that
are suitable for biotechnological applications but also fundamental research (Fig. 1). Fused to a
gene of choice, the RNA thermometer could induce protein overexpression simply by raising the
ambient temperature (Fig. 1a). The avoidance of chemical inducers reduces potential toxic effects
and lowers the overall costs when planned as a large scale application, e.g. industrial
fermentation. Experiments with the Synechocystis thermometer suggest that cooling-down the
culture will result in immediate translational shut-off. To avoid translational read-through at low
temperature, the thermosensor can be placed under additional control of environment-responsive
promoter sequences (Fig. 1b). This would combine early design strategies adopted for establishing
inducible expression systems (e.g. the LacR/O, TetR/O and AraC/I1-I2 regulatory elements, (76))
with novel translational regulator elements and, by this, allow fine-tuning of synthetic systems via
dual control. The pBAD-lacZ or pBAD-bgaB vectors used in our laboratory (27, 36) follow this
principle. Custom-designed thermometer variants could be applied to express enzymes or
activator proteins, which in turn could induce a specific cellular pathway. Two classes of synthetic
thermometer variants might be appropriate: T (Temperature)-variants, designed as psychrophilic or
thermophilic thermometers (full expression at very low or very high temperatures) and I (Induction)-
variants, with adjustable induction capacities (for example: strong, medium and weak base pairing
of SD and anti-SD sequence) for modulated expression of a certain gene product. The E. coli cspA
thermosensor is an example of a natural cold-shock sensing RNA (41, 42) which is more efficiently
translated at low temperatures. The Thermosynechococcus elongatus hspA 5‘-UTR might act as a
high-temperature sensor with an optimum at 55°C (77). These and other natural RNA
thermometers could be used for the design of synthetic T-variants. It was straightforward to
construct I-variants by rational site-directed mutagenesis of the Synechocystis hsp17 leader (this
work). The biotech engineer could choose from this toolbox and freely combine them. The I-
variants are also suitable for gradual down-regulation of selected genes (Fig. 1c). Conditional cold-
inducible knockdown strains for essential microbial genes could be generated by simply placing
the target gene under the control of synthetic thermometer I-variants and exchanging the resident
F. Discussion
19
copy. Combination of an engineered thermometer with other functional RNA domains (e.g. Rho-
independent terminators), could generate completely new RNA switches which control transcription
termination (Fig. 1d). Transcriptional control is common in riboswitches (20) but has not yet been
discovered in RNA thermometers.
Figure 1. Possible applications of synthetic RNA-based thermosensors. (A) The basic thermosensor model and (B-D) device design strategies for useful implementation of RNA thermometers as
biotechnological tools. (B) The synthetic thermometer as a temperature-sensitive link between an environment-responsive promoter (e.g. arabinose inducible) and the desired open reading frame, intended
for combined transcriptional and translational regulation of protein synthesis. (C) A regulatory RNA module, consisting of an exchangeable RNA thermometer module for differential down regulation of 3‘-attached
genes. Integration into the chromosome is possible via upstream and downstream flanking sequences. (D) Thermosensitive 5‘-element used to prematurely terminate the transcription of downstream genes.
Formation of the terminator hairpin structure is only possible under fixed temperature conditions, otherwise transcription will proceed. SD, Shine Dalgarno; ΔT, temperature shift
A
B
C
D
SD
OFF ON
ΔT
thermometer gene of choicepromoter
low medium high
complementarity
anti-SD
up downthermometer
Induction (I)-variantstarget gene
termination
UUUUU
elongationUUUUU
ΔT
+ +
F. Discussion
20
Linking engineered RNA regulatory components with existing modules could be used to construct
RNA-based biosensors which sense certain stimuli and generate stimulus-specific functional
responses (56). As it has become possible to generate fully functional and reproducing bacterial
cells by a chemically synthesized genome (78), the next steps might include customized cell-
specific organelles for the production and delivery of drugs to humans and domesticated animals
(79). Microbes engineered with optimized biosynthesis circuits might be used to convert biomass
into biofuels (80). The coupling of a sensory module to an engineered gene circuit was already
possible and allowed E. coli cells to act as biosensors that respond to input signals in a
programmable fashion (81). As „the simplest system is one in which control is exerted solely by a
direct sensor of temperature (82)“, synthetic thermometers would be the perfect supplement to
existing regulatory devices.
4. What it takes to be an RNA thermometer - the consensus algorithm
The implementation of bioinformatics into our classic experimental approach for the design of
synthetic RNA thermosensors seemed to be dispensable. However, computational approaches
might be applicable to facilitate the interpretation of experimental results. In our case, the
RNAsubopt (83) program helped us to interpret the various structure probing signals.
Implementation of in vitro and in vivo data, as obtained in this work, into mathematical algorithms
will help to accelerate the development of appropriate computational tools (84).
An appropriate iterative approach is conceivable for the SURIBA (Structures of
Untranslated Regions in Bacteria) database, which predicts putative thermosensor structures in 5‘-
UTRs preceding annotated genes (85). SURIBA uses the RNAfold program (65) for RNA structure
predictions which allows to fix certain structural parameters via dot-bracket annotation. By this, it
was possible to filter out many candidates which share certain structural features: being ROSE-like
or a FourU RNA thermometer (36, 85). Both searches were based on parameters provided by
multiple experiments (26, 27, 38, 39). Today, there are several examples of functional RNA
thermometers with highly diverse structural features (this work; Cimdins unpublished data;
Krajewski unpublished data). Novel bioinformatic tools like RNAalifold (86) could help to generate
a thermometer consensus pattern. RNAalifold scores a combination of sequence consensus and
secondary structure consensus to compute an „universal“ RNA thermometer type (Fig. 2). For this
F. Discussion
21
purpose, we used the SD-harbouring stems of six experimentally validated RNA thermometers.
Although some thermometers consist of several different hairpins that contribute to the
thermosensing capacity (in a „zipper-like“ fashion; 87), the thermometer activity per se resides in
the hairpin with the SD and anti-SD regions. This was supported by good functionality of reduced
ROSE and FourU thermometer variants (27, 39). We did not use the Listeria prfA thermometer
sequence, as the prfA SD-harbouring stem is much longer than all the others (see introduction).
Furthermore, prfA is the only described RNA thermometer from a gram-positive microbe. On the
basis of a consensus sequence (Fig. 2A), RNAalifold combines two algorithms, the Wuchty (83)
and the McCaskill (88) algorithm, to compute secondary structure predictions which illustrate the
likelihood of each base position by colour coding (Fig. 2B). The consensus RNA thermometer
could be used as input query to search for structures with both similar sequences and architecture.
The recently developed Infernal software (INFERence of RNA ALignment; 89) should be an
appropriate search tool, when combined with the SURIBA database. By this, it might be possible to
discover previously undetected RNA thermometer candidates. But, what could we learn from the
consensus pattern? What does it take to be an RNA thermometer?
Figure 2. Prediction of an RNA thermometer consensus format. (A) The SD-harbouring stems of six well-investigated RNA thermometers were used to predict a combined sequence via RNAalifold. (B) Prediction of secondary structure consensus on basis of the underlying structural alignment. Likelihood of temperature-dependent base positioning was calculated (28 and 42°C). The colour coding indicates the
probability of base positioning. blue (0), low probability; red (1), high probability; SD, Shine Dalgarno
!G: -6.96 kcal/mol!G: -4.21 kcal/mol
B
28°C 42°C
SD SD
>ROSE ( )AGGA
> ( )AGGA
> ( )AGGA
> ( )AGAG
> ( )AGGA
> ( )AGGA
consesus sequence RNAalifold:AGGA
hspA B. japonicum
agsA S. typhimurium
hsp17 Synechocystis PCC.6803
toxT V. cholerae
lcrF Y. pseudotuberculosis
ibpA E. coli
CUCGUACCCAUCUUGCUCCUUGG UUUGGCUAUG
GUUGAACUUUUGAAUAGUGAUUC GGUUAAUGAUG
GUAAUCAAUUCCUUCCACACAUC GUUAACAUUAUG
UCGAUUUUAAGAUAUAUCUUC UAGAACGCAAUG
AUAAAUAUAUACAAGUUUUAGAUUUUU CAGUAUAACAUUUAUG
CAUUCUCGCUGAUUUC GCUAUUGAUUAUG
CUAAUUCUAUGCUAUCUCGAGAC GUUAACAAUG
AA B
consensus sequence RNAalifold:
F. Discussion
22
The consensus RNA thermometer exhibits a certain degree of non-perfect base pairing
between the SD and the anti-SD, which is most probably very important for a proper temperature-
mediated melting of the structure. No less crucial are the bulged or unpaired nucleotides in close
vicinity to the SD site. The nucleobases which form an internal loop at 42°C are already imperfectly
matched at 30°C (highlighted in green, which indicates a certain tendency to seek for a new
position within the structure, Fig. 2b). They appear to function as the primary sensing sites, as they
respond already to low temperature variations and might destabilize the overall structure before
the SD sequence is released at higher temperatures, similar to the micro-ROSE thermometer (39).
Formation of the internal loop at 42°C did not affect the nearest flanking nucleotides, but the A-U
base pairs at both ends of the stem. The effect that single conformational changes within a
thermometer molecule are transmitted to distant, not directly connected parts, was revealed by
recent NMR spectroscopy experiments. Long-range effects in the Salmonella FourU thermometer
might be transduced by the surrounding hydrate shell (Dr. Jörg Rinnenthal, Schwalbe lab,
Frankfurt, personal communication) and could be further analyzed via Thz (Terrahertz) laser-
spectroscopy (in collaboration with Prof. Martina Havenith-Neven, Bochum).
5. Destabilization of a bipartite stem - a novel principle among cyanobacterial RNA thermometers?
The Synechocystis hsp17 5‘-leader, the hitherto simplest natural RNA thermosensor, has been
useful to study the effects of conformational changes that do not directly affect the SD or anti-SD
sequence. We could show in this work, that deletion of the kinked internal loop led to a „repressed“
thermometer. Recent imino exchange NMR spectroscopy data (collaboration with Prof. Harald
Schwalbe, Frankfurt) suggests that melting of the hsp17 thermometer comprises consecutive
destabilization of the bipartite stem structure (Fig. 3; by Dominic Wagner, Schwalbe lab, Frankfurt).
Helical stability decreases with increasing temperature, thus raising the probability of a nucleobase
to flip out and become solvent accessible. Therefore, imino protons exchange with water protons,
which in turn lead to attenuation of the imino resonance. Using 1D-1H NMR-spectra recorded
within the CD-derived melting range (this work), we got first insights into which base pairs open at
given temperatures during the melting process. Imino resonances from stem I (Fig. 3, highlighted
F. Discussion
23
in red) of the hsp17 5‘-UTR WT-hairpin decreased, while the resonances of G28 and G29 in stem
II (Fig. 3, highlighted in blue) became stronger at elevated temperatures. Thermally induced local
opening and closing events in stem I probably lead to conformational heterogeneity with a
stabilizing effect on stem II, and therefore explain the observed changes in intensity. Above 288 K
(14,85 °C), the resonances lost intensity, indicating the start of the melting process, which in turn is
in good agreement with the CD-derived melting curves of the hsp17 WT thermometer (this work).
This data suggests that unfolding of the hsp17 thermometer element covers a broad temperature
range and is based on independent helical unwinding of both stems, which is a novel principle of
thermometer opening.
6. Cyotherm - a novel family of „green“ RNA thermometers
We performed a multiple sequence alignment and found 26 putative cyanobacterial RNA
thermometers which might share a hsp17-like melting mechanism (77). Two 5‘-UTRs of Anabaena
variabilis heat shock genes (ava4812 and ava3076) exhibited thermometer-like activity (6-8 fold
induction of translation initiation upon temperature upshift) when tested in our established E. coli
reporter gene system (Cimdins and Klinkert 2010, unpublished data). As for hsp17, there is only
Figure 3. Temperature-dependence of the hsp17 5‘-leader imino resonances. Thermometer function is crucially based on independent and consecutive destabilization of stem I (highlighted in red ) and II
(highlighted in blue). Nucleotides of interest are marked with arrows. Temperature is indicated in Kelvin (K). Figure by Dominic Wagner (Frankfurt), revised.
G
F. Discussion
24
one imperfectly paired nucleotide within the SD sequence of these thermometers. The free energy
of the thermodynamic ensemble (SD and anti-SD) was determined for hsp17, ava4812, ava3076
and the Salmonella agsA via RNAcofold (86, 90; Fig. 4). Strikingly, it would cost 2.6 (Anabaena
ava4812/3076) to 3-fold (Synechocystis hsp17) more energy to open the cyanobacterial SD
regions than that of the FourU (Salmonella agsA) thermometer. This might explain invention of a
novel melting principle which is based on preceding reduction of the overall stability. Interestingly,
different order of the otherwise identical cyanobacterial base pair sets results in different overall
stability (-3.17 kcal/mol for the Anabaena ensemble compared to -3.63 kcal/mol for the
Synechocystis ensemble). This effect might probably be due to base stacking interactions. The
Synechocystis 5‘-GG-3‘ stack is perfectly canonically paired with the opposing 3‘-CC-5‘ stack,
which is not the case for the Anabaena 5‘-GG-3‘ stack.
As both, the Anabaena and the Synechocystis thermosensor, include internal loops, we
could define that as a common feature of cyanobacterial RNA thermometers. As it is a new RNA
thermometer family, we suggest the name Cyotherm, for Cyanobacterial thermometer. Another
cyanobacterial RNA thermosensor, the putative thermophilic Thermosynechococcus elongatus
hspA thermometer, is most probably based on a more sophisticated temperature-sensing
Figure 4. Comparative inspection of Cyotherm and FourU thermometer SD, anti-SD ensembles. Non-
canonical base pairs are marked by boxes. Free energy of the SD, anti-SD ensembles was determined with
RNAcofold (Vienna Package; Bernhart et al. 2006).
Synechocystis sp. PCC6803hsp17
Salmonella typhimuriumagsA
Anabaena variabilisava4812 / ava3076
F. Discussion
25
mechanism (77). The 5‘-UTR includes an additional G-C-rich hairpin, which might act as a
„thermobuffer“ to guarantee proper function under high temperature conditions (55 to 65°C).
Recent work focuses on development of an appropriate reporter gene system based on a
hyperthermophilic Thermus thermophilus strain.
Cyotherm elements fulfill a regulatory task not different from common thermometers. They
fine-tune expression of small heat shock genes. In contrast to previously described RNA
thermometers, these 5‘-UTRs derive from microbes which naturally risk exposure to damaging
high light intensities as cyanobacteria utilize energy from sunlight to drive photosynthesis. The
cyanobacterial light-harvesting antenna systems are comprised of supramolecular
phycobiliproteins and phycobilisomes located on the cytoplasmic surface of the thylakoid
membrane (91). The incoming light energy gathered by phycobilisomes is subsequently converted
into chemical energy by photosystem I and II (PSI and PSII). Electrons are transferred from water
to NADP+ in a cooperative manner of these two membrane-bound multisubunit complexes (92).
Both the light-driven oxidation of water catalyzed by PSII and the multiple light-driven oxidation and
reduction reactions occurring at PSI exhibit cell-damaging potential caused by generation of
reactive oxygen species (ROS) (93). The continuous impairment of PSI and II in combination with
destabilisation of the surrounding thylakoid membrane results in high turnover rates of the entire
photosystem. External influences and stresses (e.g. heat and high light stress) increase
disintegrative effects with direct impact on the microbial metabolism (94). For this reason, present
photosynthesis condition is a hallmark of the present cellular state, making it a sensible indicator to
analyze physiological effects.
7. High light-sensing by a heat shock RNA thermometer? How co-translational protein insertion
might contribute to it
The Vígh lab published several scientific essays about the thylakoid membrane being a primary
sensor of heat and UV stress, whose alteration could induce hsp17 transcription and whose
stability is increased upon subsequent insertion of the Hsp17 protein (95-100). Hsp17 is assumed
to interact with membrane lipids and might directly be involved in repair of defective photosystems
(99). We were able to manipulate lightstress-induced synthesis of Hsp17 by manipulating
functionality of the hsp17 thermometer element (this work). There is no doubt that heat shock is
F. Discussion
26
sensed by an RNA thermometer, but it is questionable if energy dissipation from light-stressed cells
could induce thermometer melting. We suggest a model, in which membrane-associated hsp17
Cyotherm RNA requires a trans-acting factor (protein or sRNA) under high light stress conditions
(Fig. 5). Co-translational insertion of Hsp17 protein into the thylakoid membrane might facilitate
rapid protection of the photosynthetic machinery. A membrane-bound, permanently translatable
hsp17 mRNA, like the Cyotherm derep variant, would cause enhanced and stress-independent
thylakoid stability as observed in the Synechocystis Derep strain (this work). Reduction in
membrane flexibility might hinder fast exchange of damaged PS components in the Derep strain.
Figure 5 is based on a model for co-translational assembly of the D1 protein into PSII (101). A
Figure 5. Hypothetical model in which hsp17 mRNA partitions between the cytoplasm and the thylakoids to preserve membrane integrity by co-translational insertion of the Hsp17 protein under stress situations. (A) Membrane-tethered hsp17 mRNA allows quick insertion of the elongating Hsp17 protein into the thylakoid membrane. The hsp17 5‘-Cyotherm element enables ribosome-binding under heat-
stress conditions (42°C). The nascent polypeptide chain complexed with ribosomes targets Hsp17 to the stromal thylakoids. Resulting changes in the protein to lipid ratio lead to enhanced membrane stability. (B) An
unknown highlight (HL)-induced regulatory factor binds to the hsp17 anti-SD site which facilitates thermometer-independent translation initiation of the small heat shock protein. SD, Shine Dalgarno
A
B
F. Discussion
27
thylakoid-associated protein that tethers mRNA to the membrane has been described (102).
Furthermore, RNA binding proteins are involved in PSII repair cycles of the eukaryotic green alga
Chlamydomonas reinhardtii (103). An AT-rich imperfect inverted repeat (ACAAgcAAATTTagTTGT)
upstream of the hsp17 homologue Thermosynechococcus vulcanus hspA exhibits protein-binding
activity (104). Nevertheless, this region is absent in hsp17 5’-UTR (105). One possibility is that
hsp17 Cyotherm activity is modulated by a small regulatory RNA under high light conditions. So
far, the asRNA IsrR is the only RNA factor known to regulate a photosynthetic component (106).
However, ongoing deep sequencing studies of cyanobacterial transcriptoms in several labs will
certainly yield further candidate sRNAs with putative influence on photosynthesis. A possible
approach to detect regulatory transcripts that bind hsp17 messenger RNA could include isolation of
stressed and non-stressed Synechocystis thylakoid membranes, extraction of the respective total
RNA and analysis via deep sequencing.
8. RNA elements as antibacterial drug targets?
Since the primary discovery of post-transcriptional gene silencing (PTGS) in plants (107) it took
about a decade to fully understand and use this natural mechanism for biotechnological
applications (108-110). 20 years later, scientists from the Berlin Charité hospital tested the potential
of using E. coli cells as vehicles to deliver interfering short hairpin RNAs into cancer cells to stop
proliferation, a procedure called „transkingdom RNA interference“ (111). This underlines the rising
possibilities in using RNA as a valuable drug, but is this omnipresent molecule also a worthwhile
target for drug development?
At the first glance, RNA might not seem very promising due to the limited number of
building blocks. It is made of only four different planar bases bound to a negatively charged
backbone. On the contrary, RNA is able to form complex 3D structures with pockets and cavities
where small molecules can bind and compete with the final steps of RNA folding (112). In theory, it
is possible to modulate the expression levels of any protein by targeting its mRNA instead of
directly inhibiting the protein. We know today that aminoglycosides developed by soil bacteria and
fungi to combat bacterial enemies (113) target ribosomal RNA rather than proteins (114). From this
perspective, RNA is an alternative to available antibiotic compounds and a solution for increasing
multi drug resistance (MDR) caused by excessive use of antibiotics in human medicine and animal
F. Discussion
28
food production. Furthermore, recent studies revealed that available antibiotics disrupt the function
of only four microbial life processes and drug development produced only one new chemical
scaffold in the past 30 years (115). Targeting RNA instead of proteins enables selective inhibition,
exploitation of multivalent interactions and by this more facile attacks on MDR (112).
A straightforward project was initiated by Ron Breaker (Yale University). To achieve
specificity, he selected RNAs that have evolved to form structured and highly selective receptors
for small drug-like metabolites (19). Professor Breaker is cofounder of BioRelix, a biotechnology
company that is pursuing the development of antibiotics that target riboswitches
(www.biorelix.com). At least 20 distinct classes of riboswitches have been reported since the first
riboswitch was reported in 2002 (19, 116-118), revealing their widespread distribution and utility in
bacteria (20). The members of each class share a highly conserved sequence and secondary
structure and bind the same metabolite. It became evident that several known antibacterial
compounds function by targeting riboswitches, e.g. the lysine analog L-aminoethylcysteine (AEC;
119) that inhibits growth of Gram-positive bacteria binds to the lysine riboswitch from the lysC gene
of Bacillus subtilis (120). In most cases, metabolite binding to the riboswitch receptor domain
represses the expression of the genes encoded by the messenger RNA. Recent results indicated
that rationally designed metabolite analogs could be used to inactivate a flavinmononucleotide
(FMN) riboswitch (121).
How are the chances to use RNA thermometers as antibacterial drug targets? Besides the
already described Listeria prfA thermosensor (28), the Vibrio toxT element (this work) and the Y.
pseudotuberculosis lcrF 5‘-UTR there is evidence that other pathogenic microbes, Brucella
melitensis, Salmonella typhimurium, Pseudomonas aeruginosa and Bacillus anthracis, might
potentially utilize RNA thermometers for posttranscriptional gene control (27; Krajewski, Kortmann
and Cimdins, unpublished data). The 4.0 Mbp genome of a pandemic V. cholerae strain is
composed of two circular chromosomes that are predicted to encode a total of 3.885 genes (122),
while the Y. pseudotuberculosis genome is 4.84 Mbp long and contains approximately 4238 genes
(123). The simple substitution of only two nucleotides in thermometer regions of Vibrio and Yersinia
genomes, respectively, resulted in drastic attenuation of virulence in both pathogenic
microorganisms (Fig. 6). The corresponding in vivo mouse experiments where conducted in
F. Discussion
29
collaboration with Prof. Karl Klose (San Antonio, USA) on Vibrio toxT (this work) and Prof. Petra
Dersch (Braunschweig, Germany) on Yersinia lcrF (this work, data not shown). Therefore, we have
strong evidence that targeting RNA thermometers with appropriate drugs could potentially mimic a
genetic knock-out.
B
com
pete
tive
inde
x
U5C-U7CWT
A
Figure 6. Introduction of single base substitutions at chromosomal thermometer sequences attenuated virulence of highly pathogenic Y. pseudotuberculosis and V. cholerae strains. (A) Point
mutations UU -28/-27 CC that enhance pairing of lcrf SD and anti-SD thermometer sites (for structure information, see supplementary data) were put on Y. pseudotuberculosis chromosome. Yersinia cells were
subsequently assayed for the ability to infect infant mice. 80% of treated mice survived at least 14 days after infection. (B) A similar result was obtained by modulating the SD site of the toxT virulence master regulator.
Stabilization of the FourU thermometer module hindered Vibrio from colonizing infant mice intestines as non of the U5C-U7C mutant cells were recovered from the intestine. WT, V. cholera wildtype strain. In vivo animal
assays were conducted in collaboration with Prof. Petra Dersch (Braunschweig, Germany) and Prof.Karl Klose (Texas, USA). All experiments used at least five mice per treatment group.
Y. pseudotuberculosismouse-survival experiment
V. choleraemouse-colonization experiment
F. Discussion
30
9. Anti-thermometer drugs - a promising attempt?
Riboswitch-antibiotics are highly specific due to the customized ligand-binding pocket of their
target molecule. Anti-riboswitch drugs might turn out to become broad-spectrum antibiotics as
riboswitches are common in many bacteria. Given that RNA thermometers are ligand-free modules
that solely respond to temperature alterations it is probably less likely to generate a compound that
effectively prohibits melting of all known RNA thermometer classes. A conceivable strategy could
be to prevent access of the ribosome to the melted thermometer at host body temperature. One
might envisage a small RNA (sRNA)-based agent, as some sRNAs mask the SD site of target
mRNAs to prevent 30S subunit binding. Because SD sites are not present in eukaryotes, a sRNA-
drug should not interfere with the mammalian metabolism. There is no strict necessity that efficient
SD-sequestration by sRNAs needs complementary base pairing with the target-SD site. The
Salmonella sRNA GcvB specifically inhibits translation of 7 distinct target mRNAs (7). Aside from
directly masking the SD sequence, GcvB can also effectively repress translation by binding to
target mRNAs at upstream sites, outside the SD site. A screen for appropriate thermometer-
targeting sRNAs could be managed by using a novel E. coli reporter system developed in the lab
of Jörg Vogel (124). The vector-based system allows examination of sRNA capacity to
downregulate a gfp-tagged target mRNA. We successfully adapted the corresponding „target-
plasmid“ (pXG-10) in this work to examine control of thermometer-mediated GFP expression by
the hsp17 Cyotherm element.
10. Custom-made drugs to target specific thermometer motifs
Not all regulatory RNAs in a modern cell can be considered relics from an early RNA world. It
appears that some biochemical functions could be well suited for RNA to carry out, and therefore
could have emerged more recently in bacterial evolution. Is it possible that some thermometer
classes are extremely ancient (most likely the heat shock sensing thermometers, as heat has
challenged microbes since their appearance on earth 3.5 billion years ago), while others are of a
more recent origin? Eric Westhof stated that RNA motifs appear to be like Russian dolls, with
smaller motifs associated into larger motifs (125). Do virulence thermometers posses certain
watermark-motifs which might help to identify them and which could be targeted by custom-made
F. Discussion
31
drugs? The toxT thermometer top portion module might, from the evolutionary perspective, be a
younger add-on to an ancient heat shock-sensing FourU thermosensor. Deletion of the toxT leader
top portion element restored a fully functional, classical heat shock RNA thermometer. The
resulting toxT-M5 variant does not only perfectly mimic thermometer function but also shares
noticeable, nearly perfect structure homology to the recently discovered E. coli htrA thermometer
(Cimdins, unpublished data; Fig. 7). We proposed that a Vibrio-specific factor (VSF) binds to the
top module and by this enables toxT thermometer function at host body temperature. If the binding
site were constantly blocked by an antagonizing drug, this would probably freeze the toxT-leader in
its untranslatable state. An alternative approach would be to inactivate the VSF.
Figure 7. Comparative secondary structure predictions of V. cholerae toxT leader variant M5 and the E. coli htrA 5‘-UTR. In silico prediction of structure and overall free energy was realized with mfold (Zuker,
2003). SD, Shine Dalgarno; 4U, FourU anti-SD region
!G : -3.1 kcal/molA B,
30
5'
5'3'
20
10
20
3'
SD4U
SD4U
35
V. cholera toxT-M5 E. coli htrA
AB
V. cholerae toxT-M5 E. coli htrA
A B
F. Discussion
32
G. Summary
Temperature belongs to the many physical and chemical parameters that free-living microbes need
to monitor in their constantly changing environment. There are numerous ways to register a
sudden change in temperature. Most of them, however, do not measure the ambient temperature
but detect indirect consequences of a temperature shift, e.g. the accumulation of misfolded
proteins after a heat shock. RNA thermometers are direct temperature sensors that fine-tune
bacterial heat shock and virulence gene expression by temperature-dependent sequestration of
the Shine Dalgarno (SD) sequence. In this study, we were able to generate a functional synthetic
RNA thermosensor and characterize two natural thermometers found in the cyanobacterium
Synechocystis sp. PCC6803 and the human pathogen Vibrio cholerae.
The main results on each of these projects are briefly summarized below:
1. Generation of synthetic RNA-based thermosensors
The presence of structurally diverse thermosensors upstream of bacterial heat shock and virulence
genes encouraged us to design a synthetic RNA thermometer by a combination of computer-based
rational design and in vivo screening. It took only two rounds of selection to obtain a synthetic
RNA thermometer that was as efficient as natural thermometers. Structure probing experiments
revealed conformational changes in the synthetic element along a temperature gradient.
2. Translation on demand by a simple cyanobacterial RNA-based thermosensor
Our systematic search for RNA thermometers in the 5‘-UTR of bacterial heat shock genes
predicted a promising, exceptionally short candidate upstream of the Synechocystis sp. PCC6803
heat shock gene hsp17. We proved hsp17 thermometer function by a newly established GFP
(Green Fluorescent Protein)-based reporter gene system. A UCCU sequence sequesters the SD
sequence (AGGA) more tightly than in other thermometers. Mutational analysis, enzymatic and
chemical structure probing and toeprinting experiments demonstrated that a large top loop, the
asymmetric internal loop and multiple A-U pairs compensate for the high thermodynamic stability of
the SD region. Chromosomal integration of repressing and derepressing point mutations in the
hsp17 thermometer resulted in Synechocystis strains with severe phenotypic defects under heat
G. Summary
33
shock and high light conditions. Our results demonstrate the physiological relevance of an RNA
thermometer in the synthesis of appropriate amounts of chaperones both in the on- and off-phase
of the heat shock response.
3. Temperature-mediated translational regulation by the V. cholerae toxT 5‘ mRNA leader
A new FourU RNA thermometer is positioned in the V. cholerae toxT leader sequence. ToxT is a
transcriptional activator that regulates genes encoding the cholera toxin and toxin-coregulated
pilus, two factors that are essential for Vibrio virulence. Translational fusions to the toxT leader
sequence resulted in reduced expression at 37°C in E. coli, which is just the opposite from what
one would expect from a typical RNA thermometer. Consistent with this finding, ribosome loading
onto the toxT transcript was efficient at 30°C but impaired at 37°C in toeprinting experiments.
Structure probing analysis of different point mutations suggested that translation inhibition at 37°C
resulted from nucleotide slippage and subsequent pseudoknot formation between two palindromic
sequences. The repressive feature of the toxT 5‘-UTR is overcome by a yet unidentified
mechanism in V. cholerae strains. Here, translation is tuned on at host body temperature (37°C).
We propose that a Vibrio-specific trans-active factor blocks the conformational switch when ToxT is
needed. Chromosomal integration of repressing point mutations in the toxT thermometer resulted
in drastic attenuation of Vibrio virulence. Our findings suggest that the toxT thermometer
contributes to Vibrio pathogenicity by modulating virulence gene expression at different stages of
host infection.
G. Summary
34
H. Zusammenfassung
Frei lebende Bakterien sind häufig den zum Teil stark fluktuierenden, physiko-chemischen
Bedingungen ihres Milieus ausgesetzt. Unter diesem Druck entwickelten sie im Laufe der
Evolution ein komplexes Überwachungssystem, welches ihnen ermöglicht, zelluläre Prozesse an
die jeweiligen Umweltparameter anzupassen. Neben dem Nährstoffangebot, der Osmolarität, dem
pH-Wert und diversen anderen Faktoren, ist vor allem die Temperatur eine für Mikroorganismen
existentielle Messgröße. Dabei bieten sich dem Bakterium zahlreiche Möglichkeiten einen
Temperaturanstieg zu registrieren, wobei die meisten intrazellulären Mess-Systeme nicht die
Temperaturerhöhung an sich, sondern deren indirekte Konsequenz, z.B. die Akkumulation falsch-
gefalteter Proteine, wahrnehmen. RNA-Thermometer hingegen sind direkte Temperatur-Sensoren,
welche die Expression bakterieller Hitzeschock- und Virulenzgene durch Temperatur-abhängige
Maskierung der Shine Dalgarno (SD) Sequenz steuern. Innerhalb dieser Arbeit war es uns
möglich, einen funktionalen, vollständig synthetischen RNA-Thermosensor zu generieren, sowie
die Charakterisierung zweier natürlicher RNA-Thermometer aus dem Cyanobakterium
Synechocystis sp. PCC6803 und dem Humanpathogen Vibrio cholerae durchzuführen.
Die Hauptergebnisse können wie folgt zusammengefasst werden:
1. Erstellung eines synthetischen RNA-basierten Thermosensors
Das Auftreten strukturell sehr unterschiedlicher Thermosensoren stromaufwärts bakterieller
Hitzeschock- und Virulenzgene hat uns veranlasst, mittels Computer-basiertem, rationalem Design
und in vivo Screening ein synthetisches RNA-Thermometer zu generieren. Nach lediglich zwei
Selektionsrunden konnten wir ein synthetisches Thermometer isolieren, welches genauso effizient
funktionierte wie natürlich vorkommende RNA-Thermometer. Die Temperatur-abhängige
Konformationsänderung des synthetischen Thermometers wurde mit Hilfe von Strukturkartierungs-
Experimenten aufgeklärt.
2. Ein cyanobakterielles RNA-Thermometer ermöglicht „Translation nach Bedarf“
Durch die systematische in silico-Suche nach RNA-Thermometern in der 5‘-untranslatierten
Region bakterieller Hitzeschockgene wurde ein extrem kleines RNA-Thermometer stromaufwärts
H. Zusammenfassung
35
des Synechocystis sp. PCC6803 Hitzeschockgens hsp17 vorhergesagt. Die hsp17-
Thermometerfunktion konnte mittels eines neu-etablierten GFP (Green Fluorescent Protein)-
basierten Reportergensystems nachgewiesen werden. Die SD-Sequenz (AGGA) liegt in diesem
Thermometer viel stärker gepaart vor, als es in anderen RNA-Thermometern der Fall ist. Durch
das Einbringen von Mutationen, enzymatischen und chemischen Strukturkartierungs-Analysen und
Toeprinting-Experimenten konnte gezeigt werden, dass ein großer Stemloop, ein asymetrischer
interner Loop und mehrere A-U-Paare die hohe thermodynamische Stabilität der SD-Region
kompensieren. Die chromosomale Integration von reprimierenden und de-reprimierenden
Punktmutationen in das hsp17 Thermometer resultierte in deutlichen phänotypischen Defekten
unter Hitzestress und Starklicht-Bedingungen. Unsere Ergebnisse demonstrieren die
physiologische Relevanz eines RNA-Thermometers in der Regulation der Chaperon-Synthese
während und nach der Hitzestress-Antwort.
3. Temperatur-abhängige Translationsregulation durch die Vibrio cholerae toxT 5‘-UTR
Ein neues FourU-Thermometer befindet sich stromaufwärts des V. cholerae toxT-Gens. ToxT ist
ein Transkriptionsaktivator, welcher die Gene für das Cholera Toxin und den Toxin-coregulierten
Pilus reguliert, beides essentielle Faktoren der Vibrio-Virulenz. Translationale Fusionen an das
toxT-Thermometer resultierten in einer reduzierten Expression bei 37°C in E. coli. In
Übereinstimmung mit diesem Ergebnis konnten isolierte Ribosomen in Toeprinting-Experimenten
zwar bei 30°C an das toxT-Transkript binden, nicht jedoch bei 37°C. Strukturkartierungs-Analysen
verschiedener punktmutierter toxT-Varianten suggerieren, dass die Inhibition der Translation bei
37°C auf die Ausbildung eines Pseudoknotens zwischen zwei palindromischen Sequenzen
zurückzuführen ist. Die reprimierende Eigenschaft des toxT-Thermometers wird in V. cholerae
selbst durch einen bislang unbekannten Mechanismus außer Kraft gesetzt. Hier erfolgt die
Initiation der Translation bei 37°C (der Körpertemperatur des menschlichen Wirts). Es ist denkbar,
dass ein Vibrio-spezifischer, trans-aktiver Faktor den konformationalen Schalter blockiert, wenn der
ToxT Aktivator benötigt wird. Die chromosomale Integration reprimierender Punktmutationen in das
toxT-Thermometer führten zu einer drastischen Attenuation der V. cholerae-Virulenz. Vermutlich
moduliert das toxT-Thermometer die Expression von Virulenzgenen in den verschiedenen Stufen
der Wirtsinfektion.
H. Zusammenfassung
36
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J. Conference contributions
1. Posters
L.C. Gaubig, J. Kortmann, T. Waldminghaus, F. Narberhaus (2007). Enterobacterial RNA thermometers. Kick-off Meeting Sensory and Regulatory RNAs in Prokaryotes (DFG priority program SPP1258), Bochum, Germany, Book of abstracts p.7 and International Symposium on RNA Ligand Interactions (SFB 579), Frankfurt a.M., Germany, Book of abstracts p.47.
J. Kortmann, F. Narberhaus (2008). Control of Vibrio cholerae virulence gene expression by an RNA thermometer. Progress Meeting Sensory and Regulatory RNAs in Prokaryotes (DFG priority program SPP1258), Kassel, Germany, Book of abstracts p.48.
J. Kortmann, S.D. Sczodrok, F. Narberhaus (2009). Control of Synechocystis heat shock gene expression by an RNA thermometer. Jahrestagung der Vereinigung für Allgemeine und Angewandte Mikrobiologie (VAAM), Bochum, Germany, Book of abstracts (BIOspektrum) p.185.
J. Kortmann, F. Narberhaus (2009). A novel RNA thermometer controls Vibrio cholerae virulence gene expression. Metabolism Meets Virulence (International Symposium on Metabolism and Bacterial Pathogenesis), Munich, Germany, Book of abstracts p.113.
J. Kortmann, S.D. Sczodrok, F. Narberhaus (2009). A novel RNA thermometer controls expression of small heat shock genes in Synechocystis. International Meeting Sensory and Regulatory RNAs in Prokaryotes (DFG priority program SPP1258), Berlin, Germany, Book of abstracts PP 30.
J. Kortmann, F. Narberhaus (2009). Adjusting Virulence: A novel RNA thermometer controls Vibrio cholerae virulence gene expression. International Meeting Sensory and Regulatory RNAs in Prokaryotes (DFG priority program SPP1258), Berlin, Germany, Book of abstracts PP 31.
K. Böhme, A.K. Heroven, R. Steinmann, W. Opitz, J. Kortmann, F. Narberhaus, P. Dersch (2010). The synthesis of important virulence factors in Yersinia pseudotuberculosis underlies the control of a complex regulatory network during the infection process. Jahrestagung der Vereinigung für Allgemeine und Angewandte Mikrobiologie (VAAM), Kassel, Germany, Book of abstracts (BIOspektrum) p.116.
J. Conference contributions
45
J. Kortmann, S.D. Sczodrok, F. Narberhaus (2010). Translation on demand: A simple cyanobacterial RNA-based thermosensor is critical for stress management. Jahrestagung der Vereinigung für Allgemeine und Angewandte Mikrobiologie (VAAM), Kassel, Germany, Book of abstracts (BIOspektrum) p.123.
K. Böhme, R. Steinmann, J. Kortmann, A.K. Heroven, F. Narberhaus, P. Dersch (2010). Regulatory RNAs coordinate expression of important virulence factors in Yersinia pseudotuberculosis. Kick-off Meeting Sensory and Regulatory RNAs in Prokaryotes (DFG priority program SPP1258), Kassel, Germany, Book of abstracts p.40.
2. Talks
J. Kortmann, F. Narberhaus (2008). Adjusting Virulence: RNA thermometers in pathogens. Jahrestagung der Vereinigung für Allgemeine und Angewandte Mikrobiologie (VAAM), Frankfurt a.M., Germany, Book of abstracts (BIOspektrum) p.82.
J. Kortmann, S.D. Sczodrok, J. Rinnenthal, H. Schwalbe, F. Narberhaus (2010). Translation on demand by a simple cyanobacterial RNA-based thermosensor. Jacques Monod at 100 - Commemorative Symposium (Institut Pasteur), Paris, France, no book of abstracts.
J. Kortmann, S.D. Sczodrok, J. Rinnenthal, H. Schwalbe, F. Narberhaus (2010). Translation on demand: A simple cyanobacterial RNA-based thermosensor is critical for stress management. Kick-off Meeting Sensory and Regulatory RNAs in Prokaryotes (DFG priority program SPP1258), Kassel, Germany, Book of abstracts p.24.
J. Conference contributions
46
K. Appendix
1. Curriculum vitae
Dipl.-Biol. Jens Frank Kortmann
Date of birth: 25.03.1980Nationality: GermanFamily status: not married
Scientific education
2001 - 2003 Elementary studies in biology, Ruhr-University Bochum
2003 - 2005 Main studies in biology, Ruhr-University Bochum
2005 - 2006 Diploma project („Molekulare Analyse der Interaktion von Nac2 und RBP40, zweier posttranskriptioneller Regulatoren der plastidären Genexpression in Chlamydomonas reinhardtii”) at the Department of General and Molecular botany (Prof. Dr. Jörg Nickelsen), Bochum
since 2007 PhD project (“Structure and function of novel RNA thermometers”) at the Department of Microbial biology (Prof. Dr. Franz Narberhaus), Bochum
Fellowships
2007 - today PhD fellowship from the “Studienstiftung des Deutschen Volkes”
2009 FEMS supported young scientist (FEMS young scientists meeting grant)
Other relevant activities
2009 - 2010 -Doctoral representative of the Ruhr-University Research School -Board member of the Research School Executive Board -Organisation committee: Science College, Section Day, Business College
Publications2007 “Synthesis of the D2 protein of photosystem II in Chlamydomonas is
controlled by a high molecular mass complex containing the RNA stabilization factor Nac2 and the transcriptional activator RBP40.” (Schwarz C, Elles I, Kortmann J, Piotrowski M, Nickelsen J) Plant Cell
2008 “Generation of synthetic RNA-based thermosensors.” (Waldminghaus T, Kortmann J, Gesing S, Narberhaus F) Biol Chem
2010 “Translation on demand by a simple RNA-based thermosensor” (Kortmann J, Sczodrok S, Rinnenthal J, Schwalbe H, Narberhaus F) submitted
K. Appendix
47
2. Erklärung
Hiermit erkläre ich, dass ich die Arbeit selbstständig verfasst und bei keiner anderen Fakultät
eingereicht und dass ich keine anderen als die angegebenen Hilfsmittel verwendet habe. Es
handelt sich bei der heute von mir eingereichten Dissertation um sechs in Wort und Bild völlig
übereinstimmende Exemplare.
Weiterhin erkläre ich, dass digitale Abbildungen nur die originalen Daten enthalten und in keinem
Fall inhaltsverändernde Bildbearbeitung vorgenommen wurde.
Bochum, den
(Jens Kortmann)
K. Appendix
48
L. Eigenanteil an den Publikationen (Manuskripten)
„Struktur und Funktion neuartiger RNA-Thermometer“
Dissertation, Jens Frank Kortmann, Lehrstuhl für Biologie der MikroorganismenReferent: Prof. Dr. Franz NarberhausKorreferent: Prof. Dr. Ulrich KückBochum, den 15. Oktober 2010
1. Waldminghaus, T., Kortmann, J., Gesing, S. and Narberhaus, F. (2008) Generation of synthetic RNA-based thermosensors. Biol. Chem., 389, 1319-1326.
30% Planung, Durchführung und Auswertung der Experimente; 25% Verfassen des Manuskripts
2. Kortmann, J., Sczodrok, S., Rinnenthal, J., Schwalbe, H. and Narberhaus, F. (2010) Translation on demand by a simple RNA-based thermosensor. submitted.
90% Planung; 70% Durchführung und 80% Auswertung der Experimente; 100% Verfassen des Manuskripts
3. Kortmann, J., Weber, G., Klose, KE. and Narberhaus, F. (2010) Temperature-mediated translational regulation by the Vibrio cholerae toxT 5‘ mRNA leader is based on nucleotide slippage and pseudoknot formation. in preparation.
100% Planung; 90% Durchführung und 90% Auswertung der Experimente; 100% Verfassen des Manuskripts
L. Eigenanteil an den Publikationen
49