Processing and turn-over of small non-coding RNA OxyS in E. coli & post-transcriptional regulation of RpoS levels by small non-coding RNAs OxyS and DsrA and the Hfq protein in E. coli [Elektronische Ressource] / vorgelegt von Sobha Rani Basineni

Basineni, Sobha Rani

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2010
• Nombre de lectures: 29
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Processing and turn-over of small non-
coding RNA OxyS in E .coli
&
Post-transcriptional regulation of RpoS
levels by small non-coding RNAs OxyS and
DsrA and the Hfq protein in E.coli


Inaugural-Dissertation
Zur Erlangung
Des
Doktorgrades der Naturwissenschaften
(Dr. rer. Nat)


vorgelegt von
M. Sc. - Biol. Sobha Rani Basineni
Aus Mudigubba (Andhrapradesh, India)


angefertigt am Institut für Mikrobiologie und Molekularbiologie
Fachbereich Biologie und Chemie
Justus-Liebig-Universität Gießen




Giessen, August 2010




Die vorliegende Arbeit wurde angefertigt am Institut für Mickrobiologie und Molekularbiologie des
fachbereiches 08 der Justus-Liebig-Universität Giessen in der zeit von Juni 2007 bis August 2010 unter der
Leitung von Prof. Dr. Gabriele Klug.

















1. Gutachterin: Prof. Dr. Gabriele Klug
Institute für Mikrobiologie und Molekularbiologie
Justus-Liebig- Universität Giessen

2. Gutachterin: Prof. Dr. Annegret Wilde
Institute für Mikrobiologie und Molekularbiologie
Justus-Liebig-Universität Giessen

















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My beloved Parents
And
MMMMyyyy bbbbeeeelllloooovvvveeeedddd SSSSiiiisssstttteeeerrrrssss aaaannnndddd BBBBrrrrooootttthhhheeeerrrrssss

Contents

Publications I
1. Introduction 1
1.1 Discovery of sRNAs in bact e..................................................ria ................................................... ............... 1
1.2 Regulatory roles of RN As ........................ 3
1.3 Role of Hfq in sRNA func tion .................. 4
1.4 Regulation of RpoS Translat ion .............. 5
1.4.1 DsrA, a translational activator of RpoS ............................................. 6
1.4.2 OxyS, a negative regulator of RpoS translation .................................................. ................................ 6
1.5 Consequences of ncRNA/mRNA pair ing . 7
1.5.1 Translation inhibiti o..................................................n ................................................... ...................... 8
1.5.2 Translation activation ..................... 8
1.5.3 Coupled degradation of ncRNA/mRNA duplex ................................. 9
1.6 The role of Ribonucleases in Post-Transcriptional Regu lation ............... 10
1.6.1 Endoribonucleas es ........................ 10
1.6.2 Exoribonucleas es ........................... 11
1.7 Polyadenylation and Poly (A)-mediated d ecay ...................................... 12
1.8 The Role of RNases in small Non-Coding RNA proce ..................................................ssing ...................... 13
1.9 Objectives of this wo ..................................................rk ................................................... ......................... 15
2 Materials 1 6
2.1 Chemicals and Reagen ts ....................... 16
2.2 Antibiotic s ............................................. 17
2.3 Plasmid s ................................................ 17
2.4 Oligonucleotide s ................................... 17
2.5 Bacterial Stra i..................................................ns ................................................... .................................... 18
2.6 Radioactive nucleotides used for labe ling .............................................. 18
2.7 Enzyme s ................................................ 18
2.8 Molecular weight standar ds ................ 19
2.9 Molecular biological reagents and kits 19
2.10 Antibodie s ........................................... 19
2.11 Equipment and devic es ....................... 20
3 Methods 2 1
3.1 E.coli cultivat io..................................................n ................................................... ................................... 21
3.1.1 E.coli plating culture .......................... 21
3.1.2 E.coli liquid cu lture ............................ 21
0
3.1.3 Preparation of glycerol stocks for thCe s-t8r0ain collectio n.................................................. ............. 21
Page I
Contents

3.2 Plasmid minipreparation by alkaline ly..................................................sis .............................................. 21
3.3 Chromosomal DNA isolation from E. coli ................................................. 22
3.4 Gel electrophoresis of D NA ................................................... .................. 23
3.4.1 Gel extractio n: ................................... 23
3.5 Molecular cloni ng ................................. 23
3.5.1 Polymerase chain reaction (PCR) ...... 23
3.6 Preparation of E.coli competent cells for electropo r..................................................ation ..................... 24
3.6.1 Transformation by electroporat ion .. 24
3.7 Extraction, purification and analysis of mRNA from................................................... E.coli .................... 25
3.7.1 RNA isolat io..................................................n ..................................... 25
3.7.2 Northern B lot ..................................... 26
3.8 SDS-polyacrylamide gel electrophore sis ................................................. 29
3.9 Western Blo t ......................................... 30
3.10 Transcription inhibiti on ................................................... ...................... 31
3.11 Translation inhibitio n ......................... 31
3.12 Oxidative Stre ss .................................. 32
4 RESULTS 3 3
4.1 Effect of growth rate on OxyS turn-over in E. coli M..................................................G1655 ................... 33
4.2 The effect of the RNA chaperone, Hfq on OxyS s tability ........................ 34
4.3 Influence of the endoribonucleases RNase E and RNase III on OxyS tu........................................ rnover 35
4.4 Influence of the exoribonucleases PNPase, RNase II and endoribonuclease RNase E on OxyS 39t urnover
4.5 The influence of DsrA on the decay rate of OxyS .................................... 41
4.6 The decay rate of OxyS in double mutants – Nh3fq43,1 BL321hfq, N3431dsrA, BL321dsrA ......... 43
4.7 The effect of growth phase on the stability of the rpo S mRNA .............. 47
4.8 The effect of hydrogen peroxide and growth phase on OxyS, DsrA and RpoS levels in an E. coli wild
type strain ..................................................................................................... ................................................... 51
4.9 Hfq affects the levels of RpoS protein and of OxyS and DsrA ................ 55
4.10 Effect of DsrA on the levels of RpoS protein and on Ox y..................................................S levels .......... 57
4.12 Hfq, DsrA and OxyS influence turn-over of RpoS .................................. 59
5 Discussion 6 2
5.1 Growth phase dependent turn-over of OxyS sRNA ................................ 62
5.2 Influence of endo- and exoribonucleases on the turn-over of OxyS ....... 64
5.3 Role of Hfq on the turn-over o f.................................................. OxyS ................................................... ... 66
Page II
Contents

5.4 Role of DsrA on the turn-over o f.................................................. OxyS ................................................... . 67
5.5 Growth phase dependent turn-over of rpoS mRNA ................................ 68
5.6 Influence of OxyS, DsrA and Hfq on RpoS s ynthesis ............................... 69
6 Summary 7 3
7 Zusammenfassung 7 4
8 References 7 5
9 Supplementary data 91
9.1 Expression analyses of OxyS, and DsrA sRNAs under oxidative stress condition in all studied E.coli
strains...................................................................................................... ......................................................... 91
9.2 Expression analysis of RpoS, OxyS and rpoS mRNA under oxidative stress condition in E.coli MG1655::
∆hfq, ∆dsrA .................................................. ................................................. 92
9.3 Stability determination of RpoS in E.coli MG1655 lacking Hfq and DsrA under oxidative stress in late-
exponential and in stationary growth pha ..................................................se ............................................... 93
9.4 Stability determination of OxyS in E.coli MG1655 and E.coli MG1655∆rpoS, under oxidative stress in
early exponential and in stationary growth ph ase ...................................... 94
+
9.5 Stability determination of OxyS in E.coli MG1655 & strain MG1655∆hfq and E.co)l i &N 3433 (rne
ts
N3431 (rne) under oxidative stress in early exponential and in stationary growth phase without
transcription inhibition ................................................... ............................... 95
9.6 Stability determination of rpoS mRNA in E.coli MG1655 and strain MG1655∆hfq, under oxidative stress
in early exponential and