A green fluorescent protein (GFP) based approach to study translational control in Escherichia coli [Elektronische Ressource] : establishment and applications / vorgelegt von Johannes Urban

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A green fluorescent protein (GFP) based approach to study translational control in Escherichia coli: Establishment and applications Den Naturwissenschaftlichen Fakultäten der Friedrich-Alexander-Universität Erlangen- Nürnberg zur Erlangung des Doktorgrades vorgelegt von Johannes Urban aus Nürnberg Als Dissertation genehmigt von den Naturwissenschaftlichen Fakultäten der Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 20.02.2008 Vorsitzender der Promotionskommission: Dr. E. Bänsch Erstberichterstatter: Prof. Dr. Hillen Zweitberichterstatter: PD Dr. Wilde Index ZUSAMMENFASSUNG.....................................................................................................4 SUMMARY ........................................................................................................................5 1. INTRODUCTION ...........................................................................................................6 1.1 Non-coding RNA-mediated gene regulation in bacteria .........................................................6 1.1.1 Translational repression and activation of gene expression..................................................7 1.1.2 Interference with protein function.........................................................................................10 1.
Publié le : mardi 1 janvier 2008
Lecture(s) : 47
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Source : WWW.OPUS.UB.UNI-ERLANGEN.DE/OPUS/VOLLTEXTE/2008/898/PDF/JOHANNESURBANDISSERTATION.PDF
Nombre de pages : 132
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A green fluorescent protein (GFP)
based approach to study translational control in
Escherichia coli:
Establishment and applications







Den Naturwissenschaftlichen Fakultäten
der Friedrich-Alexander-Universität Erlangen-
Nürnberg
zur
Erlangung des Doktorgrades







vorgelegt von
Johannes Urban

aus Nürnberg



Als Dissertation genehmigt
von den Naturwissenschaftlichen Fakultäten
der Universität Erlangen-Nürnberg






























Tag der mündlichen Prüfung: 20.02.2008

Vorsitzender der Promotionskommission: Dr. E. Bänsch

Erstberichterstatter: Prof. Dr. Hillen

Zweitberichterstatter: PD Dr. Wilde


Index
ZUSAMMENFASSUNG.....................................................................................................4

SUMMARY ........................................................................................................................5

1. INTRODUCTION ...........................................................................................................6
1.1 Non-coding RNA-mediated gene regulation in bacteria .........................................................6
1.1.1 Translational repression and activation of gene expression..................................................7
1.1.2 Interference with protein function.........................................................................................10
1.2 Protein factors involved in sRNA-mediated gene regulation ...............................................11
1.2.1 The RNA-chaperone Hfq .....................................................................................................11
1.2.2 Ribonucleases......................................................................................................................12
1.3 Identification of sRNAs and their targets ...............................................................................13
1.3.1 Approaches to identify non-coding RNAs ............................................................................13
1.3.2 Approaches to identify targets of non-coding RNAs............................................................16
1.3.3 Target verification ................................................................................................................19
1.4 Aim of the study ........................................................................................................................20

2. RESULTS....................................................................................................................21
2.1 Establishment of a GFP-based two-plasmid reporter system..............................................21
2.1.1 Principle of the system.........................................................................................................21
2.1.2 Design and application of specialized reporter plasmids.....................................................23
2.1.2.1 The standard gfp fusion vector pXG-10 24
2.1.2.2 The RACE gfp fusion vector pXG-20 28
2.1.2.3 The operon gfp fusion vector pXG-30 31
2.1.3 High specificity of regulatory RNA/RNA interactions revealed by the GFP system ............34
2.1.4 E. coli as suitable host to assess heterologous RNA/RNA interactions ..............................39
2.1.5 Transfer of the GFP-based reporter system into different genetic backgrounds.................41
2.1.5.1 The important role of Hfq in sRNA-mediated posttranscriptional regulation 41
2.1.5.2 Efficient sRNA-mediated translational silencing in the absence of a functional RNA
degradosome 42
2.1.6 Regulatory RNA/RNA interactions monitored in individual cells..........................................43
2.1.7 A uniform approach allows quick chromosomal integration of reporter cassettes ..............45
2.1.8 Improved reporter gene activity using the new superfolder GFP variant ............................48
2.2 Identification of novel sRNA targets in E. coli .......................................................................50
2.2.1 Computer-based sRNA target predictions ...........................................................................50
2.2.2 Identification and verification of non-coding RNA targets using proteomics .......................52

I Index
2.3 The sRNA-mediated posttranscriptional regulation of E. coli glmS ................................... 54
2.3.1 The sRNA SroF activates expression of glucosamine-6-phosphate synthase GlmS......... 54
2.3.2 GlmS accumulation upon SroF overexpression is independent of yfhK............................. 56
2.3.3 SroF mediates discoordinate expression of the dicistronic glmUS mRNA to enhance GlmS
synthesis and is accompanied by processing of the glmUS mRNA ............................................ 58
2.3.4 Sequence elements located in the glmUS IGR are sufficient for SroF dependent activation
of GlmS synthesis ........................................................................................................................ 60
542.3.5 SroF and a putative σ dependent promoter are conserved in enterobacteria.................. 61
2.3.6 Hfq strongly binds the glmUS IGR in vitro and is required for activation in vivo................. 63
2.3.7 Loss of yhbJ promotes GlmS accumulation and can be suppressed by deletion of the non-
coding RNA sraJ........................................................................................................................... 65
2.3.8 SroF and SraJ are related RNAs and act hierarchically to activate GlmS synthesis.......... 67
2.3.9 SraJ acts directly on the glmS mRNA to enhance translation by preventing formation of an
inhibitory mRNA structure ............................................................................................................ 71
2.3.10 GlmS accumulates in pcnB mutant strains in an sroF and sraJ dependent manner........ 78
2.3.11 Loss of SroF polyadenylation in pcnB mutant strains leads to stabilization of the sRNA
and results in activation of GlmS synthesis via SraJ.................................................................... 80

3. DISCUSSION ..............................................................................................................83
3.1 The GFP-based two-plasmid reporter system....................................................................... 83
3.1.1 GFP as reliable reporter of sRNA-mediated translational regulation.................................. 83
3.1.2 Hfq but not the degradosome contributes to sRNA-mediated translational control............ 87
3.2 Identification of novel sRNA targets in E. coli....................................................................... 89
3.3 Two sRNAs use different mechanisms to enhance translation of the glmS mRNA.......... 90
3.3.1 Multiple factors are involved in the posttranscriptional regulation of E. coli glmS ............. 90
3.3.2 SraJ in concert with Hfq is the direct activator of glmS translation ..................................... 91
3.3.3 SroF indirectely activates GlmS synthesis by antagonizing the decay of SraJ .................. 93
3.3.4 Loss of SroF polyadenylation causes GlmS overproduction in pcnB mutant cells............. 95
3.3.5 The posttranscriptional regulation of GlmS expression ...................................................... 96

4. MATERIAL AND METHODS.......................................................................................98
4.1 Material ...................................................................................................................................... 98
4.2 Media and growth conditions................................................................................................ 102
4.3 Bacterial strains and plasmids.............................................................................................. 102
4.4 Methods ................................................................................................................................... 108
4.4.1 RNA isolation and Northern blot analysis.......................................................................... 108
II Index
4.4.2 In vitro transcription and 5´ end labeling of RNA ...............................................................110
4.4.3 Gel mobility shift assays ....................................................................................................110
4.4.4 Enzymatical and chemical cleavage of RNA in vitro..........................................................111
4.4.5 Primer extension ................................................................................................................112
4.4.6 cDNA synthesis and cloning for RNA 3´ end sequencing .................................................112
4.4.7 Rapid amplification of cDNA ends (5´ RACE)....................................................................113
4.4.8 In vivo whole-cell colony plate fluorescence imaging ........................................................114
4.4.9 Liquid culture whole-cell fluorescence measurements ......................................................114
4.4.10 Fluorescence measurements in microtiter plates ............................................................115
4.4.11 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)...................................................116
4.4.12 Western blot analysis.......................................................................................................116
4.4.13 In vitro translation.............................................................................................................117

5. REFERENCES ..........................................................................................................118

6. ABBREVIATION INDEX............................................................................................126

PUBLICATIONS AND PRESENTATIONS ....................................................................128

DANKSAGUNG.............................................................................................................129


III
Zusammenfassung

Nicht-kodierende kleine RNAs (sRNAs) sind zentrale Regulatoren bakterieller
Genexpression und wirken oftmals, indem sie über direkte RNA/RNA Interaktionen mit
den leader Regionen trans-kodierter mRNAs deren Translation inhibieren oder aktivieren.
Da immer mehr Transkripte der Klasse der sRNAs zugeordnet werden, aber die
regulatorische Funktion der meisten noch unbekannt ist, wurde eine Methode etabliert, die
es erlaubt, sRNA vermittelte Translationskontrolle in vivo nachzuweisen und zu studieren.
Basierend auf dem grün-fluoreszierenden Protein (GFP) wurde ein zwei-Plasmid
Reportersystem in E. coli entwickelt, um das regulatorische Potential von sRNA/mRNA
Paaren aufzuklären. Hierbei werden sRNA und mögliche mRNA Zielmoleküle konstitutiv
von kompatiblen Plasmiden in der gleichen Zelle überexprimiert, was zur weitgehenden
Abkopplung vom chromosomalen transkriptionellen Netzwerk führt. Mögliche mRNA
Zielregionen, die den 5´ untranslatierten Bereich und einige der amino-terminal
kodierenden Aminosäuren enthalten, werden translational an gfp fusioniert. Spezielle
Reporterplasmide erlauben es, neben konventioneller Klonierung auch cDNA primärer
Transkripte über ein modifiziertes 5´ RACE Protokoll oder intra-operonische
Zielsequenzen polycistronischer mRNAs in ein Mini-Operon als duales Reporterkonstrukt
zu klonieren. Mit Hilfe dieses Systems wurde erstmalig eine Vielzahl gut charakterisierter
E. coli sRNA/mRNA Paare einheitlich untersucht. Translationale Regulation wurde
zuverlässig über Koloniefluoreszens, Fluoreszensmessung von Flüssigkulturen bei
Wachstum unter Standardbedingungen oder in Mikrotiter-Platten, aber auch in
individuellen Zellen mittels Durchflusszytometrie beobachtet. Darüberhinaus wurden
sRNA/mRNA Interaktionen verwandter Bakterien, wie z.B. Vibrio Spezies, und der
Einfluss diverser Proteine auf die sRNA vermittelte Genregulation untersucht.
Zusätzlich wurden mit Hilfe des GFP-basierten Reportersystems bisher unbekannte sRNA
Zielgene bestätigt, die in dieser Arbeit durch bioinformatische Vorhersagen oder
Proteomanalysen identifiziert wurden. Dabei wurde der molekulare Mechanismus genauer
untersucht, durch den die beiden sRNAs SroF und SraJ die Translation der E. coli glmS
mRNA aktivieren, die für ein essentielles Enzym des Aminozucker-Metabolismus kodiert.
Obwohl beide sRNAs sehr große Ähnlichkeit in Sequenz und Struktur aufweisen,
aktivieren sie die GlmS Synthese auf unterschiedliche Weise. SraJ verhindert die Bildung
einer cis-inhibierenden mRNA Struktur durch direkte Basenpaarung mit dem leader der
glmS mRNA und aktiviert so deren Translation. Im Gegensatz hierzu stimuliert SroF die
GlmS Synthese indirekt, indem sie die Inaktiverung von SraJ verhindert. Eine
vergleichbare hierarchische sRNA Kaskade, die zur posttrankriptionellen Aktivierung der
Genexpression führt, wurde bisher noch nicht beschrieben.
4
Summary

Small noncoding RNAs (sRNAs) are an emerging class of regulators of bacterial gene
expression, which mainly modulate translation of trans-encoded target mRNAs by physical
base-pairing to mRNA leader squences. The here presented work contributes to
overcome one of the current limtations in sRNA research, the lack of assigned function for
most sRNAs, by providing a novel tool to study sRNA-mediated translational control and
target mRNA recognition in vivo.
A two-plasmid system, based on the green fluorescent protein (GFP) as reporter of gene
expression, was established to rapidly monitor the regulatory potential of sRNA/target
mRNA pairs under investigation in E. coli. Herein, sRNA and potential mRNA targets are
constitutively co-expressed at high levels from compatible plasmids within the same cell,
resulting in the widely uncoupling from the chromosomal transcriptional network. Putative
target mRNA regions, covering the 5´ full-length untranslated region and the first amino-
terminal coding residues are translationally fused to gfp, upon insertion into specialized
reporter plasmids. This vector series allows, besides conventional directed cloning, cDNA
cloning of primary mRNA transcripts by an adapted 5´ RACE strategy, and cloning of
intra-operonic target regions derived from polycistronic mRNAs into a dual-reporter mini-
operon. Using this system, multiple well-defined E. coli sRNA/target mRNA pairs have
been investigated for the first time in a uniform manner in vivo. Faithful target regulation
was observed by colony fluorescence imaging, by fluorometric measurements upon
growth in standard laboratory cultures or in microtiter plates, or on the single cell level by
fluorescence microscopy and flow cytometry. Moreover, sRNA/target mRNA interactions
of distantly related bacteria, e.g. Vibrio species, and the contribution of protein factors to
sRNA-mediated target regulation were investigated.
In addition, the here presented GFP-based reporter system proved useful to verify
previously unknown sRNA targets in E. coli, which have been predicted or identified by
proteomic approaches in this study. In this regard, the molecular mechanisms by which
two sRNAs, namely SroF and SraJ, activate the translation of the E. coli glmS mRNA,
coding for an essential function in amino sugar metabolism, was investigated in detail.
Although both sRNAs are closely related in sequence and structure, they were found to
use a distinct mechanism to activated GlmS synthesis. While direct base-pairing of SraJ to
the glmS mRNA leader results in enhanced translation by preventing the formation of a
self-inhibitory mRNA hairpin, SroF indirectly enhances GlmS synthesis by antagonizing
the decay of the SraJ transcript. This is the first example of two sRNAs which act in a
hierarchical cascade to activate gene expression by a posttranscriptional mechanism.
5 1. Introduction
1. Introduction

Gene regulation is a prerequisite for all living organisms to adapt to their specific habitats.
The precise timing of gene expression is required to respond to fluctuations in the
availability of nutrients, pH, temperature and other environmental changes. In bacteria, as
well as in eukaryotic organisms, severel layers of gene regulation exist to realize this goal.
Considered to be the most important means in this regard is the transcriptional regulation
of gene expression, which involves transcription factors that either activate or repress
promoter activity and thereby adjust mRNA synthesis to the required levels. The use of
alternative sigma factors in bacterial RNA-polymerase holoenzymes enables the
orchestrated expression of regulons at the level of promoter recognition. Besides other
transcriptional mechanisms, such as DNA bending or premature transcription termination,
posttranslational regulation provides another widespread regulatory layer. Hereby, protein
activity might be altered by proteolytic cleavage, posttranslational modification, or effector
molecule binding. In addition, a wealth of regulatory functions have been found to be
carried out at the posttranscriptional level over the last decades. It is now established that
RNA is not only a passive messenger in the cell, determined to serve as a blueprint for
protein synthesis, but can itself execute regulatory functions.

1.1 Non-coding RNA-mediated gene regulation in bacteria

Posttranscriptional gene regulation is widespread in all kingdoms of life. It may act in ´cis´,
implying that the regulatory function is encoded in the same DNA locus or even within the
same mRNA molecule which is about to be affected. Natural riboswitches are members of
the latter group and are mainly located in the 5´ untranslated region (UTR) of mRNAs.
They provide flexible RNA platforms that may adopt different structures upon binding of
ligands or temperature shift, which in turn induce alterations in the accessibility of
ribosome binding sites (RBS) or affect mRNA stability (Winkler and Breaker, 2005). On
the other hand, polyadenylation of the 3´ ends of certain mRNA species leads to
destabilization by promoting rapid degradation via 3´ - 5´ exonucleases (Kushner, 2002).
Another group of regulatory RNAs comprises the cis-encoded antisense RNAs of
plasmids and phages generally located on the opposite strand of the target gene on which
they act. This physical location brings about undisrupted sequence complementarity to the
target mRNA, and well described examples of regulation include control of plasmid copy
number, postsegregational killing and the replication cycle of bacteriophages (Wagner et
al., 2002). Trans-encoded sRNAs have emerged in recent years as yet another class of
regulatory RNAs and are the focus of the present study. Typically, these molecules are
6 1. Introduction
50-200 nucleotides (nt) in size and do not contain expressed open reading frames
(ORFs). In Escherichia coli (E. coli), about 70 members of this group have been identified
to date (Vogel and Sharma, 2005) and most of them are transcribed from independent
sRNA genes located in intergenic regions (IGRs) which contain Rho-independent
transcription terminators. Regulation of gene expression by sRNAs is predominantly
mediated by physical sRNA/target mRNA interactions that are based on short and
imperfect complementarity.
1.1.1 Translational repression and activation of gene expression

Only a small portion of the identified sRNAs in E. coli has been assigned to well
characterized functions while for the majority the targets they regulate are still unknown.
Under laboratory conditions, many sRNAs are induced when cells enter stationary phase
or when certain stress conditions such as cold shock, sugar stress, acid stress, membrane
stress or iron depletion are sensed. This observation, and the described examples,
suggests that most sRNAs are involved in stress response regulation (Wagner and
Darfeuille 2006).
Base-pairing of sRNAs most often occurs in the 5´UTR of the target mRNA and is likely
supported by the bacterial Sm-like protein Hfq, which will be discussed in later sections.

Translational repression
The most common mode of described sRNA action so far in E. coli is the inhibition of
translation of the targeted mRNA. In these cases, binding occurs in proximity to the Shine-
Dalgarno (SD) sequence, preceding the translational start codon. This ribosome binding
site is complementary to the 3´ end of the 16S RNA of the ribosome and is thought to
ensure effective formation of the ribosome-mRNA complex by base-pairing, helping to
initiate translation (Shine and Dalgarno, 1974). MicF, the first reported chromosomally
encoded trans-acting antisense RNA in E. coli, was shown to inhibit translation of an
mRNA (ompF mRNA - coding for the outer-membrane protein F) by complementary
binding within the 5´UTR (Andersen and Delihas, 1990). Stable duplex formation that
covers RBS and translational start codon of the ompF mRNA blocks ribosome entry and
results in low levels of the OmpF porin. Other major outer-membrane proteins in E. coli
are regulated by sRNAs that exploit similar mechanisms. In this regard, the MicC and
MicA sRNAs target the 5´UTRs of ompC and ompA mRNAs, respectively (Chen et al.,
2004; Udekwu et al., 2005), to inhibit translation.
Blocking of SD sequences (Fig. 1.1) seems not to be the only way in which sRNAs acting
on 5´UTRs can repress translation. In E. coli, the tisAB locus encodes an SOS-induced
toxin (TisB) whose translation is counteracted by binding of the sRNA IstR-1 to a region ~
7 1. Introduction
100 nt upstream of the tisB RBS under non-SOS conditions (Vogel et al., 2004). A recent
report showed that in vitro the RBS of tisB is sequestered by an intrinsic structure whereas
the region of IstR-1 binding is single stranded (Darfeuille et al., 2007). Experimental
evidence suggested this single stranded region to be a ribosome loading site, which
allows translation of tisB by standby ribosomes sliding into the transiently open translation
initiation region. Under non-SOS conditions, IstR-1 binding is thought to inhibit translation
by competing with standby ribosomes.
sRNA-mediated translational repression is not limited to the 5´UTRs of monocistronic
target mRNAs, but is also observed within the intergenic regions of polycistronic
messengers. In E. coli, the galETKM operon encodes enzymes involved in galactose
metabolism. The trans-encoded sRNA Spot42, itself transcriptionally controlled by the
cyclic adenosine monophosphate (cAMP) receptor protein Crp-cAMP system, targets
internal sequences of the galETKM mRNA. Induced in response to high glucose levels,
Spot42 masks the RBS of the galK cistron and inhibits its translation without affecting the
upstream galET cistrons (Møller et al., 2002a). The differential synthesis ratio of the UDP-
galactose-4-epimerase GalE and galactokinase GalK as mediated by Spot42 is described
as discoordinate operon expression.

Translational activation
Besides translational repression, sRNA binding to the 5´UTR of a target mRNA can also
stimulate translation. The mRNA of the rpoS gene encoding the stationary phase sigma
Sfactor σ contains an extraordinary long (565 nt) untranslated leader, and RpoS synthesis
is crucial to adapt to various stresses (Hengge-Aronis, 2002). Under non-activating
conditions, the rpoS 5´UTR folds into an inhibitory stem structure which masks the RBS
and renders the mRNA translation-incompetent. Two sRNAs, namely DsrA and RprA, can
activate translation by base-pairing with 5´UTR sequences that are located upstream of
the RBS and are involved in maintenance of the self-inhibitory structure (Lease et al.,
1998; Majdalani et al., 2002). Binding of the sRNAs opens the stem within the rpoS leader
and results in activation of translation by freeing the RBS and promoting ribosome entry
(Fig. 1.1). Although both sRNAs act by a related mechanism, involving pairing to the same
region in the rpoS mRNA, DsrA and RprA are expressed under different conditions. While
DsrA is induced at low temperatures, RprA expression peaks upon cell surface stress
which seems to be due to transcriptional regulation via the RcsC/YojN/RcsB phosphorelay
system (Repoila and Gottesman, 2001; Majdalani et al., 2002). As shown for the post-
8

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