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Identification of Small Regulatory Rnas and Their Targets in Bacteria

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Identification of Small Regulatory Rnas and Their Targets in Bacteria Identification of Small Regulatory RNAs and Their Targets in Bacteria Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Technischen Fakultat¨ der Universitat¨ Bielefeld vorgelegt von Cynthia Mira Sharma Bielefeld, im Mai 2009 Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Technischen Fakultat¨ der Universitat¨ Bielefeld. Vorgelegt von: Dipl.-Biol. Cynthia Mira Sharma Angefertigt im: Max-Planck-Institut fur¨ Infektionsbiologie, Berlin Tag der Einreichung: 04. Mai 2009 Tag der Verteidigung: 22. Juli 2009 Gutachter: Prof. Dr. Robert Giegerich, Universitat¨ Bielefeld Dr. Jorg¨ Vogel, Max-Planck-Institut fur¨ Infektionsbiologie, Berlin Prof. Dr. Wolfgang Hess, Universitat¨ Freiburg Prufungsausschuss:¨ Prof. Dr. Jens Stoye, Universitat¨ Bielefeld Prof. Dr. Robert Giegerich, Universitat¨ Bielefeld Dr. Jorg¨ Vogel, Max-Planck-Institut fur¨ Infektionsbiologie, Berlin Prof. Dr. Wolfgang Hess, Universitat¨ Freiburg Dr. Marilia Braga, Universitat¨ Bielefeld Gedruckt auf alterungsbestandigem¨ Papier nach ISO 9706. Dedicated to my parents, Ingrid Sharma and Som Deo Sharma ACKNOWLEDGEMENTS At this point, I would like to thank everybody who contributed in whatever form to this thesis. In particular, I am grateful to the following persons: . my supervisor Dr. Jorg¨ Vogel for giving me the opportunity to work in a superb scientific environment and for his continuous support and guidance throughout the past several years, . Prof. Dr. Robert Giegerich for enabling me to submit this thesis to the University of Bielefeld and for valuable suggestions during several visits to his institute, . Prof. Dr. Wolfgang Hess for his willingness to evaluate this thesis, . Dr. Fabien Darfeuille (a. k. a. the “master of the gels”) for a great stay in his lab in Bordeaux and many fruitful discussions, . Dr. Steve Hoffmann, Kai Papenfort, Dr. Titia Plantinga, and Dr. Alexandra Sittka for their contributions to this thesis, helpful discussions, and enjoyable collaborations, . Dr. Marc Rehmsmeier for initial suggestions about sRNA target predictions using RNAhybrid, . Franziska “Franntz” Seifert and Sandy Pernitzsch for technical assistance in various experiments, . Birgit Grett, unserer guten Fee“ im Labor, fur¨ ihren Beitrag zum Gelingen unserer ” Experimente, . all proofreaders of this thesis, especially Kathrin “Kathi” Frohlich¨ for her final scrutiny and Kai “Lillifee” Papenfort for his critical comments and, in addition, for a lot of fun with them in the lab, their support and help, . all my colleagues and former members of the Vogel lab for a fantastic time and kind cooperation, . last but not least my parents, my sister, and my friends for their patience and encouragement, . and, of course, Nikolaus for immensely supporting and encouraging me, especially during the past few months. U G C G A G C U A C T C T AAAA GCGC T A U A G A C A A C C G A C GG A G A C G C G G G G G U A C U G G C C CA C C G C G G C G C U G G A C G A U A U U A G C A U U A C G G C U G C G C G C G hankA U ou! CONTENTS ListofFigures ........................................ xvii ListofTables ......................................... ......... xx Abbreviations ......................................... ........ xxi Units ................................................ xxii Multiples .............................................. xxiii 1. Introduction ................................................... .. 1 2. Biological Background .............................................. 5 2.1. Regulation of gene expression by regulatory RNAs in bacteria . ......... 7 2.1.1. Translational repression . ......... 7 2.1.2. Translational activation . ....... 9 2.1.3. sRNA mediated sequestration of protein activity . 10 2.1.3.1. 6SRNA ......................................... 10 2.1.3.2. CsrB,CsrC ..................................... 11 2.1.4. Riboswitches and RNA thermosensors . ..... 12 2.1.5. Cis-endodedsRNAs ...................................... 12 2.1.6. sRNAs with dual functions . ...... 13 2.2. Additional factors involved in gene regulation by bacterial sRNAs . ......... 13 2.2.1. The Sm-like protein Hfq . 14 2.2.2. Ribonucleases .................................... ........ 15 2.3. Approaches for the identification of bacterial sRNAs . .......... 17 2.3.1. Direct Labelling . ...... 17 2.3.2. Geneticscreens ................................... ........ 18 2.3.3. Biocomputational screens . ...... 19 2.3.4. Identification of sRNAs by transcription factor binding sites . 22 2.3.5. RNomics .......................................... ..... 23 2.3.6. Homologysearches ................................. ....... 24 2.3.7. Microarray detection . ....... 25 2.3.8. Co-purification with proteins . ..... 26 x 2.4. Target identification and verification of bacterial sRNAs . .......... 26 2.4.1. Experimental approaches . ........ 27 2.4.1.1. Proteomics . 27 2.4.1.2. Microarrays .................................... 28 2.4.1.3. Co-immunoprecipitation of direct interaction partners . 28 2.4.2. Biocomputational target predictions . ..... 29 2.5. Multiple targeting by sRNAs in bacteria . ...... 31 2.6. Model pathogens used in this study . .......... 32 2.6.1. Salmonella enterica SerovarTyphimurium ....................... 32 2.6.1.1. Hfqphenotype ................................... 33 2.6.1.2. Salmonella sRNAs .................................. 33 2.6.2. Helicobacter pylori ........................................ 34 2.6.2.1. Small regulatory RNAs in H. pylori ...................... 34 3. Multiple targeting of ABC transporter mRNAs by GcvB sRNA ............... 35 3.1. Results ........................................... ........... 37 3.1.1. Characterization of GcvB sRNA in Salmonella .................... 37 3.1.2. GcvB targets dppA and oppA mRNAs in vivo and in vitro ............ 40 3.1.3. A conserved G/U-rich region mediates GcvB repression in vivo ........ 43 3.1.4. MoreGcvBtargets .................................. ...... 45 3.1.5. GcvB inhibits translation initiation in vitro ....................... 49 3.1.6. GcvB inhibits gltI translation by binding far upstream of the start codon . 51 3.1.7. The C/A-rich target site enhances gltI translation . 53 3.1.8. Creation of an upstream C/A-rich target site permits translational control of anunrelatedmRNA ....................................... 53 3.2. Discussion ........................................ ............ 53 4. GcvB RNA, a global regulator of genes involved in amino acid metabolism ...... 61 4.1. Results ........................................... ........... 62 4.1.1. Expression of GcvB from an arabinose inducible plasmid . 62 4.1.2. Microarray-based identification of GcvB target mRNAs in Salmonella . 62 4.1.3. Prediction of C/A elements reveals further targets . 68 4.1.4. Identification of additional mRNAs that contain the GcvB target site . 71 4.1.5. GcvB represses expression of the glycine transporter CycA . ........ 74 xi 4.1.6. GcvB binds to cycA mRNA in vitro ............................ 76 4.1.7. Diverse GcvB mutants indicate multiple binding sites for cycA mRNA . 77 4.1.8. GcvB inhibits translation initiation of cycA mRNA in vitro ............ 81 4.2. Discussion ........................................ ............ 82 5. Analysis of Hfq-bound RNAs in Salmonella by 454 pyrosequencing ........... 89 5.1. Results ........................................... ........... 91 5.1.1. Experimental setup for deep sequencing of Hfq-associated RNAs ....... 91 5.1.2. Biocomputational analysis of 454 data . 93 5.1.2.1. Removal of 5’ end linker sequences and poly(A) tails . 93 5.1.2.2. Read length distribution . 94 5.1.2.3. Removalofshortreads ............................ 95 5.1.2.4. Read mapping to a reference genome . 96 5.1.2.5. Overlaps to annotations . 97 5.1.2.6. Visualization of mapped sequences . 99 5.1.3. Analysis of cDNA sequencing results of Hfq-associated RNA . 100 5.1.4. Visualizing Hfq-dependent RNAs at the nucleotide level . 101 5.1.5. Hfq-dependent sRNAs are highly associated with Hfq . 103 5.1.6. Identification of expressed Salmonella sRNAs .................... 104 5.2. Discussion ........................................ ............ 109 6. Deep sequencing reveals the primary transcriptome of Helicobacter pylori ...... 113 6.1. Results ........................................... ........... 115 6.1.1. Biocomputational prediction of ncRNAs in H. pylori ................ 115 6.1.2. Depletion of processed RNAs . 121 6.1.3. Deep sequencing of Helicobacter cDNAlibraries .................. 121 6.1.4. Mapping of transcriptional start sites . 127 6.1.5. Global TSS annotation . 128 6.1.6. 454 sequencing reveals a 6S RNA homologue of H. pylori ............ 130 6.1.7. NovelsRNAs ..................................... ....... 132 6.1.8. Additional short ORFs within the H. pylori genome ................. 134 6.2. Discussion ........................................ ............ 137 xii 7. Conclusions ................................................... ... 147 8. Material and Methods .............................................. 149 8.1. Material .......................................... ............ 149 8.2. GeneralMethods ................................... ............ 153 8.2.1. Bacterial cell culture . ....... 153 8.2.1.1. Media .......................................... 153 8.2.1.2. Preparation of electrocompetent Salmonella cells . 154 8.2.1.3. Transformation of chemically competent E. coli cells . 154 8.2.1.4. Growthcurves ..................................
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