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

Jurban

Sujets

• Biologie

Informations

• Publié par: friedrich-alexander-universitat_erlangen-nurnberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 48
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

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 .............................................