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

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DISCUSSION supercoiling, and so is the gene for its phosporylation (aceK) in the same ace operon of the bypass genes. Interestingly the icdA mutants can be readily isolated by selecting for nalidixic acid resistance (gyrase inhibitor) [Helling & Kukora, 1971]. Furthermore, we found that the de novo pathway of nucleotide biosynthesis explicitly involves the Hyp genes (Figure 15B). Most of these genes have promoters with a GC-rich ‘discriminator’ sequence, which confers sensitivity to both high negative supercoiling and the regulatory nucleotide ppGpp [Neidhardt, F.C. et al., 1987; Nygaard et al., 1996]. Notably, both the superhelicity and ppGpp concentration are elevated in fis cells [Travers & Muskhelishvili, 2005b], indicating a homeostatic regulation mechanism. The observed coupling of distinct supercoiling sensitivities to essential metabolic pathways provides new insights into the mechanisms that coordinate central metabolism, and also sheds light on the puzzling observation that mutations in metabolic genes can affect DNA topology [Hardy & Cozzarelli, 2005]. 4.3 Activity of tyrT promoter - Interaction of supercoiling and FIS at the promoter level We also investigated the molecular mechanism of regulation of an individual gene transcription and an interplay between supercoiling and transcriptional regulator FIS using the tyrT promoter system as a model. The promoters of stable RNA operons are subject to a complex regulatory network which coordinates the activity of translation machinery with the metabolic demands of the cell. Our data indicate that the effects of fis on tyrT transcription in vivo are a consequence of at least two phenomena, one local and one global: a direct activation by binding of FIS to UAS and interaction with RNA polymerase at the promoter [Bokal et al., 1995; Muskhelishvili et al., 1995] and general FIS dependent reduction in the average superhelical density of the DNA template [this work and Schneider et al., 1997]. These effects would be expected, respectively, to increase and decrease tyrT transcription consistent with the homeostatic mode of regulation. Recently Rochman et al. [Rochman et al., 2002] have shown that stabilization of local 63
DISCUSSION writhe in the UAS by FIS protects rrnA P1 promoters from inactivation at sub-optimal superhelical densities. This compensatory effect is also apparent with wild-type tyrT in vivo when expressed from hyperhegatively supercoiled plasmid DNA. We see here that the mutations in the core promoter that alter the sensitivity to supercoiling also alter the response to FIS, although the UAS region containing the FIS binding sites remain intact. Also the increased supercoiling of the wild-type promoter template in the fis cells [Auner et al., 2003] may, in principle compensate for the absence of FIS. The distinct roles of core promoter and UAS in sensing the supercoiling and FIS revealed in this work, together describe the extent of regulatability built into the stringently controlled tyrT promoter. 4.3.1 Promoter structure and function We have studied the effects of the three mutations of tyrT core promoter: a change in the -35 region to the consensus sequence TTGACA, an increase in the spacing between the –10 and –35 hexamers from a suboptimal 16bp to the consensus 17bp [Lisser et al., 1993], and a 4bp substitution in the discriminator region (TTAA in place of CCCC, Figure 16B). The mutations towards the consensus exhibit substantially increased activity in vivo (Figure 17). The permanganate reactivity of promoter mutants’ show that all the core promoter mutants studied in the absence of FIS and NTPs have enhanced interactions with polymerase relative to the wild-type promoter in the TG of the extended –10 region and the –10 region itself (Figure 21A). The mutation in the spacer and –35 hexamer also result in the loss of dependence on negative superhelicity, showing that the sensitivity of the tyrT promoter to this parameter is determined by more than one aspect of sequence organization. A further similarity is that both these mutations act in vitro to enhance interactions with the –10 region (Figure 21). Notably, neither of these mutations enhanced permanganate reactivity in the vicinity of the transcription start point in the absence of NTPs. We infer that a major effect of these mutations is to promote untwisting of DNA within the –10 hexamer. The inference we draw is that a 64
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Coordinated regulation of gene expr
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Parts of this work has been publish
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ABSTRACT promoter region determines
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ACKNOWLEDGEMENTS first impression o
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TABLE OF CONTENTS 2.2.5 Preparation
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LIST OF TABLES List of tables Table
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LIST OF FIGURES List of Figures Fig
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ABBREVIATIONS Abbreviations ∆61
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ABBREVIATIONS TBE Tris TS tyrT35U t
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INTRODUCTION Salmonella: “In more
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INTRODUCTION Figure 1: Schematic il
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INTRODUCTION UP element -35 -10 Spa
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INTRODUCTION can also influence elo
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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 and 34: INTRODUCTION formed as a repercussi
Page 35 and 36: MATERIALS AND METHODS 2 Materials a
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: DISCUSSION cells maintain viability
Page 83 and 84: DISCUSSION 4.3.2 Mechanism of trans
Page 85 and 86: CONCLUSION 5 Conclusion Regulation
Page 87 and 88: REFERENCES Bordes, P., Conter, A.,
Page 89 and 90: REFERENCES Gruber, T.M., and Gross,
Page 91 and 92: REFERENCES molecular basis for sele
Page 93 and 94: REFERENCES Pan, C.Q., Finkel, S.E.,
Page 95 and 96: REFERENCES Schroder, O., and Wagner
Page 97 and 98: REFERENCES Willenbrock, H., and Uss
Page 99 and 100: APPENDIX I gene blattner ratio p-va
Page 127 and 128: APPENDIX I Supplementary table - VI
Page 129 and 130: 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 p-va
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APPENDIX I gene blattner ratio A/B
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Statement of sources Declaration I
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