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

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RESULTS Addition of FIS increased the reactivity of bases T-8 and T-11 at the wild-type promoter (lanes 1–3). Also, in the presence of FIS the permanganate reactivity for all promoter derivatives was further increased, the tyrTS and tyrT35 mutants exhibiting a greater response than tyrTD. These observations show that at all promoters, albeit to varying degrees, FIS enhances the interaction with the −10 region. The formation of initiation (open) complex at the wild-type tyrT promoter requires the presence of GTP and CTP to allow the synthesis of the dinucleotide pppGpC [Muskhelishvili et al., 1997]. Figure 21B shows that under these conditions enhanced permanganate reactivity was observed at the wild-type promoter principally at positions T+3, T+4, T+10, T+26 and addition of FIS had very small, if any, effect (lanes 1–3). The reactivity at positions +3 and +4, and possibly that at +10, corresponds to the transcription “bubble” in the DNA. The cleavage patterns observed in the mutant promoters differ from the wild-type in one major respect, permanganate cleavage being apparent in all of them in the −10 and/or discriminator regions. For all the mutant promoters FIS enhances cleavage significantly suggesting that in these cases FIS can facilitate initiation complex formation. A similar effect of FIS on the wild-type promoter has previously been observed on a linear DNA template [Muskhelishvili et al., 1997]. 57
DISCUSSION 4 Discussion The idea of this work was to integrate the effects of fis and hns genes, representing the ‘digital’ components of genetic information, and variations in DNA superhelicity forming the ‘analog’ component in order to understand the logics of concerted rearrangement of global transcription. In a review by Travers and Muskhelishvili [Travers & Muskhelishvili, 2005b], it is argued that the transcription initiation is predominantly determined by the topology of the initiation complex and the intrinsic twist of the DNA, which strongly depends on the mode of DNA packaging. This later property of the DNA is determined by the chromatin proteins. A high amount of variability is seen in DNA packaging by different chromatin proteins. FIS has been shown to stabilize both the toroidal and plectonemic forms of DNA, while H-NS stabilizes a tight plectoneme [Maurer et al., 2006; Schneider et al., 2001]. Further, repression or activation of individual gene promoters can also be a result of differential binding of chromatin proteins. H-NS is known to be a global repressor of transcription [Dorman, 2004] and manages to do so by occluding the polymerase binding site or by trapping the polymerase in abortive cycle [Schroder & Wagner, 2000]. H-NS can also silence a gene by DNA oligomerization of two intrinsically bent, AT rich, remotely placed upstream and downstream binding sites as observed in the case of bgl operon [Ueguchi & Mizuno, 1993]. FIS also represses promoters by promoter occlusion (gyrA promoter) and polymerase trapping (gyrB promoter) [Schneider et al., 1999]. Activation by FIS, a more widely studied phenomenon, is accomplished by direct interaction with RNAP and also by trapping the supercoils and directing them to facilitate promoter opening. The ability of FIS in regulating multiple steps of transcription is evident from the study of different tyrT promoter mutants in the current work, a detailed discussion of which will follow in the subsequent sections. The effects exerted by global regulators like FIS and H-NS are complex and require investigation on both global (genome) and local (single promoter) levels. However, both of these phenomena are associated with 58
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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
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 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: RESULTS contrast the tyrTD promoter
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
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
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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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