Coordinated regulation of gene expression by E ... - Jacobs University

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INTRODUCTION [Rimsky et al., 2001]. At similar superhelical density HU and H-NS can exert opposite effect on the transcription. HU binds and bends DNA resulting in a decrease in pitch angle (the angle between two adjacent major grooves), while H-NS binding leads to an increase in pitch angle. Replacing HU by H-NS on a plectonemic DNA would lead to over twisting of the DNA rendering it less transcriptionally active [Travers & Muskhelishvili, 2007]. The transcription of stable RNA genes is one of the determining elements of growth rate. The promoters of tyrT and rrnA P1 (exemplary stable RNA promoters) are an excellent model systems to fathom the intricacies of the interactions among the global regulators on a miniaturized location. The unique promoter architecture involving a GC rich discriminator confers ‘stringent response’ and ‘growth-rate control’ [Travers, 1980] imposing a need for negative superhelicity for promoter activity [Pemberton et al., 2000]. During stringent response, a result of amino acid starvation, the regulatory nucleotide ppGpp accumulates and inhibits the synthesis of rRNA operons by blocking the RNAP at the initiation step. Growth-rate control is essentially a phenomenon of balanced exponential growth controlled by a combination of promoter elements (a suboptimal spacer region and -35 hexamer sequences) and a need for UAS bound by activator protein, FIS. Binding of H-NS counteracts activation by FIS [Tippner et al., 1994]. Thus it appears that the NAPs on binding DNA exert both global and local effects. It was of interest therefore to investigate, whether the growth phasedependent patterns of transcription are associated with NAP-dependent changes in distributions of effective superhelicity. 17
MATERIALS AND METHODS 2 Materials and methods 2.1 Materials 2.1.1 Enzymes & chemicals Unless otherwise stated, chemicals were procured from Applichem GmbH and Sigma- Aldrich. Lysozyme (100 000 U/mg) Restriction & nicking enzymes Taq polymerase Reverse transcriptase: Superscript II & MLV-RT Random primers (3 μg/μl) DNase I Vaccinia Topoisomerase I RNA polymerase Cyanine 3 and Cyanine 5 dyes Merck New England Biolabs New England Biolabs Invitrogen Invitrogen Qiagen & Ambion EPICENTRE ® Biotechnologies Bohreinger Manhein & Purified in house (Geertz, M) Cy3-dCTP and Cy5-dCTP from GE healthcare UK limited. CyDye3 dCTP and CyDye5 dCTP from Amersham Biosciences UK limited. 18
Page 1 and 2: Coordinated regulation of gene expr
Page 3 and 4: Parts of this work has been publish
Page 5 and 6: ABSTRACT promoter region determines
Page 7 and 8: ACKNOWLEDGEMENTS first impression o
Page 9 and 10: TABLE OF CONTENTS 2.2.5 Preparation
Page 11 and 12: LIST OF TABLES List of tables Table
Page 13 and 14: LIST OF FIGURES List of Figures Fig
Page 15 and 16: ABBREVIATIONS Abbreviations ∆61
Page 17 and 18: ABBREVIATIONS TBE Tris TS tyrT35U t
Page 19 and 20: INTRODUCTION Salmonella: “In more
Page 21 and 22: INTRODUCTION Figure 1: Schematic il
Page 23 and 24: INTRODUCTION UP element -35 -10 Spa
Page 25 and 26: INTRODUCTION can also influence elo
Page 27 and 28: INTRODUCTION have more base-pairs t
Page 29 and 30: INTRODUCTION Figure 6: Graphical re
Page 31 and 32: INTRODUCTION shown that H-NS reache
Page 33: INTRODUCTION formed as a repercussi
Page 37 and 38: MATERIALS AND METHODS NaCl 10 gm YT
Page 39 and 40: MATERIALS AND METHODS LZ41 3 pyrF28
Page 41 and 42: MATERIALS AND METHODS 2.2 Methods 2
Page 43 and 44: MATERIALS AND METHODS given using a
Page 45 and 46: MATERIALS AND METHODS phenol: chlor
Page 47 and 48: MATERIALS AND METHODS Filtered (by
Page 49 and 50: MATERIALS AND METHODS free water (A
Page 51 and 52: RESULTS 3 Results Growth phase-depe
Page 53 and 54: RESULTS Neither the fis nor the hns
Page 55 and 56: RESULTS these identified genes (by
Page 57 and 58: RESULTS furthermore the colour of t
Page 59 and 60: RESULTS During the entire growth cy
Page 61 and 62: RESULTS transcripts of a growth pha
Page 63 and 64: RESULTS phosphorylation, are associ
Page 65 and 66: RESULTS Table 4: RT-PCR analysis of
Page 67 and 68: RESULTS Figure 16: Schematic repres
Page 69 and 70: RESULTS parC) which allows the supe
Page 71 and 72: RESULTS Figure 19: In vitro transcr
Page 73 and 74: RESULTS contrast the tyrTD promoter
Page 75 and 76: DISCUSSION 4 Discussion The idea of
Page 77 and 78: DISCUSSION In a study by Balke & Gr
Page 79 and 80: DISCUSSION cells maintain viability
Page 81 and 82: DISCUSSION writhe in the UAS by FIS
Page 83 and 84: DISCUSSION 4.3.2 Mechanism of trans
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CONCLUSION 5 Conclusion Regulation
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REFERENCES Bordes, P., Conter, A.,
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REFERENCES Gruber, T.M., and Gross,
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REFERENCES molecular basis for sele
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REFERENCES Pan, C.Q., Finkel, S.E.,
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REFERENCES Schroder, O., and Wagner
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REFERENCES Willenbrock, H., and Uss
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APPENDIX I gene blattner ratio p-va
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APPENDIX I Supplementary table - VI
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APPENDIX I fabB b2323 0.6336 0.0043
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APPENDIX I ibpA b3687 2.4960 0.0181
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APPENDIX I pepP b2908 1.2182 0.0368
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APPENDIX I speG b1584 0.4151 0.0001
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APPENDIX I ycjX b1321 4.0841 0.0141
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APPENDIX I yhfY b3382 1.4063 0.0453
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APPENDIX I gene blattner ratio A/B
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Statement of sources Declaration I
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