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Birte Reichenbach Regulation of glucosamine-6-phosphate synthase synthesis by a hierarchical acting cascade composed of two small regulatory RNAs in Escherichia coli. Dissertation zur Erlangung des Doktorgrades der Mathematisch-Natrurwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Birte Reichenbach aus Reinbek Göttingen 2009 D7 Referent: Prof. Dr. Jörg Stülke Korreferent: Prof. Dr. Botho Bowien Tag der mündlichen Prüfung: 19.10.2009 Danksagung Danksagung Zu allererst danke ich PD Dr. Boris Görke für die Bereitstellung des spannenden Themas und die Betreuung meiner Doktorarbeit. Ich habe sehr von der Möglichkeit profitiert, meine eigenen Ideen auszuprobieren. Auch die Möglichkeit jederzeit das weitere Vorgehen zu diskutieren und so auf neue Ideen zu kommen haben sehr zum Gelingen dieser Arbeit beigetragen. Außerdem möchte ich mich sehr herzlich bei Herrn Prof. Dr. Jörg Stülke für die Übernahme der Doktorvaterschaft und seine immer vorhandene Unterstützung bedanken. Desweiteren danke ich Herrn Prof. Dr. Botho Bowien für seine Unterstützung und die freundliche Übernahme des Korreferats. Für die finanzielle Unterstützung meiner Arbeit danke ich der Studienstützung des Deutschen Volkes. Ohne diese Unterstützung, wäre die Durchführung dieser Doktorarbeit unmöglich gewesen. Ich möchte außerdem allen Mitgliedern des Labors und der Abteilung danken, die zum Gelingen dieser Arbeit beigetragen haben. Insbesondere möchte ich mich bei Sabine Lentes bedanken, die mich während der letzten Phase der Arbeit besonders unterstützt hat. Auch allen Praktikanten, die ich während meiner Arbeit betreut habe, möchte ich für ihr Interesse am und teilweise sogar erfolgreichen Beiträge zum Projekt danken: Juliane Wolters, Sabine Zeides, Antje Hillmann, Kerstin Voigt, Yvonne Göpel und Tilmann Künzl. Allen Labormitgliedern, sowohl den ehemaligen als auch den derzeitigen, möchte ich außerdem für die gute Arbeitsatmosphäre und die vielen Kaffee- und Mittagspausen danken: Ricarda, Björn, Denise, Jens, Katja, Sabine, Kerstin, Yvonne, Antje und ganz besonders natürlich Kalpana. Die „Let’s have a coffee“-Pausen nach dem morgendlichen Animpfen und Gele starten vermisse ich schon jetzt. Mein besonderer Dank gilt natürlich meiner ganzen Familie. Manfred, dir möchte ich für deine Unterstützung danken. Besonders während der schwierigen Phasen der Arbeit, hast du mich überzeugt, dass es auch wieder besser werden wird und ja schon ein Ende abzusehen ist. Insbesondere danke ich auch meiner Mutter, Oma und Opa, Großmutti und Großvati, Dirk und Petra, für die Unterstützung während meines Studiums, während der Diplomarbeit und auch während der langen Zeit der Doktorarbeit. Table of contents Table of contents Danksagung ....................................................................................................... Table of contents ............................................................................................... List of abbreviations ......................................................................................... 1 Summary ......................................................................................................... 3 1. Introduction ................................................................................................. 5 1.1 Posttranscriptional regulation ..................................................................... 5 1.1.1 Riboswitches and ribozymes.............................................. 6 1.1.2 Small regulatory RNAs ....................................................... 7 1.1.3 Types of sRNAs and their preferred modes of action ........ 8 1.1.3.1 Cis -encoded antisense RNAs ............................... 8 1.1.3.2 Trans -encoded sRNAs ......................................... 8 1.1.4 Physiological functions and targets of sRNAs in E. coli ..... 9 1.1.4.1 Toxin/antitoxin systems ........................................ 9 1.1.4.2 Regulation of outer membrane proteins ............. 10 1.1.4.3 Regulation of sugar metabolism ......................... 11 1.1.4.4 Regulation of iron metabolism ............................ 13 1.1.4.5 Regulation of rpoS .............................................. 13 1.2 Control of transcription initiation .................................................... 14 1.2.1 Two-component systems ................................................. 14 1.2.2 Alternative sigma factors .................................................. 15 1.2.3 The rpoN operon .............................................................. 16 1.2.3.1 lptAB ................................................................... 16 1.2.3.2 rpoN .................................................................... 17 1.2.3.3 hpf ...................................................................... 18 1.2.3.4 ptsN and npr ....................................................... 18 1.2.3.5 yhbJ .................................................................... 19 1.3 Amino sugar metabolism in E. coli ................................................ 20 1.4 Aims of this study .......................................................................... 23 1.5 List of publications ......................................................................... 24 2. Feedback control of glucosamine-6-phosphate synthase GlmS expression depends on the small RNA GlmZ and involves the novel protein YhbJ in Escherichia coli .............................................................................................. 25 Table of contents 3. The small RNA GlmY acts upstream of the sRNA GlmZ in the activation of glmS expression and is subject to regulation by polyadenylation in Escherichia coli ................................................................................................................. 61 4. Dual control by perfectly overlapping σ54 - and σ70 -promoters adjusts small RNA GlmY expression to different environmental signals .............................. 84 5. YhbJ is involved in modulation of activity of the glmY σ54 dependent promoter in Escherichia coli ......................................................................... 119 6. Discussion................................................................................................ 143 6.1 Feedback regulation of glmS expression by the sRNA GlmZ ................ 143 6.2 A second sRNA, GlmY, also regulates glmS expression ............. 145 6.3 On the role of Hfq in the GlmY/Z-system ..................................... 146 6.4 On the role of RNase E in the GlmY/Z-system ............................ 146 6.5 Turning off the signal: Control of GlmY halflife by polyadenylation .......................................................................................................... 148 6.6 Regulation of GlmY expression by a dual promoter and the two- component system GlrK/R ................................................................. 150 6.7 What is the function of YhbJ? ...................................................... 152 6.8 How is the GlcN6P signal sensed by the cell? ............................ 155 6.9 Evolution of GlmY and GlmZ ....................................................... 157 6.10 Function of the GlrR/GlrK two component system ..................... 159 7. Conclusion and perspectives ................................................................... 162 8. References............................................................................................... 164 Curriculum vitae ........................................................................................... 185 List of abbreviations List of abbreviations 5’UTR 5’ untranslated region ABC ATP binding cassette ABS activator binding site ATP adenosine triphosphate bp base pairs DIG digoxygenin DNA deoxyribonucleic acid DTT Dithiothreitol EMSA electro mobility sihft analysis Fig. Figure FMDP N3-(4-methoxyfumaroyl)-L-2,3-diaminopropanoic acid Glc6P glucose-6-phosphate GlcN glucosamine GlcNAc N-Acetylglucosamine GlcN1P glucosamine-1-phosphate GlcN6P glucosamine-6-phosphate GlcNAc6P N-Acetylglucosamine-6-phosphate GlmS glucosamine-6-phosphate synthase GTP guanosine triphosphate IDA iodacetamide IHF integration host factor IPTG isopropyl-β-D-1-thiogalactopyranoside LB Luria Bertani LPS lipopolysaccharide mRNA messanger RNA NEM N-ethylmaleimide Neu5Ac N-acetylneuraminic acid nt nucleotide OD optical density PAP I poly(A) polymerase PCR polymerase chain reaction PNPase polynucleotide phosphorylase 1 List of abbreviations PTS phosphotransferase system PVDF polyvinylidene difluoride membrane RACE rapid amplification of cDNA ends RBS ribosomal binding site RNA ribonucleic acid rpm rounds per minute rRNA ribosomal RNA SD Shine-Dalgarno sequence SDS sodium dodecyl sulfate sRNA small RNA TAP tobacco acid pyrophosphatase TCS two component system UDP uridin diphosphate X-Gal 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside 2 Summary Summary Amino sugars are essential for the biosynthesis of the bacterial cell wall and of lipopolysaccharides. The enzyme glucosamine-6-phosphate synthase (GlmS) catalyzes the formation of glucosamine-6-phosohate (GlcN6P) from fructose- 6-phosphate and glutamine, which is the first committed step in the biosynthesis of amino sugars. In Escherichia coli , GlmS is encoded together with enzyme GlmU in the bi-cistronic glmUS operon. While the enzymatic activity of GlmU is needed all the time, GlmS is only required, when no external amino sugars are available. Due to the fact that both genes are co- transcribed, it always was a mystery how differential
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