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

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RESULTS Both ∆61 wild-type and ∆61 D constructs had low activity at low superhelical density, about tenfold higher activity at near physiological density and a lower, but intermediate, activity on hypernegatively supercoiled plasmid. However the two promoters differed in their response to varying superhelicity in the presence of the UAS. The wild-type promoter had an enhanced response to superhelicity. Again the promoter lacked significant activity at low superhelicity but was strongly activated at nearphysiological superhelical density and notably, activity was further increased with the hypernegatively supercoiled plasmid, similar to the effect observed by Bowater et al [Bowater et al., 1994]. However, with the discriminator mutant the presence UAS substantially increased promoter activity at all superhelical densities tested but did not alter the profile of the response. In sharp contrast the activities of the tyrTS and tyrT35U mutant promoters lacking the UAS were much less dependent on variations in superhelicity with both of these promoters exhibiting high activity in vivo at low superhelical density. Thus the loss of dependence on supercoiling was accompanied by a comparable loss of dependence on the UAS containing FIS binding sites. The relative activities of the promoters in this genetic background differ somewhat from those observed in CSH50 as also did the dependence of the activity of the discriminator mutant on the UAS. 3.2.4 Dependence of promoter activity on superhelical density in vitro The in vivo results on the supercoiling sensitivity of promoter constructs were verified by in vitro transcription assays with truncated wild-type and mutant promoter constructs. Transcription was performed using supercoiled and linearized templates. We observed higher levels of transcription from mutants compared to the wild-type on linearized template, while transcription of supercoiled template is comparable between the wild-type and the mutants (Figure 19). Of all the mutants, 35U shows highest activity on the linear template and is also least sensitive to changes of supercoiling as seen in the in vivo experiments (Figure 18). 53
RESULTS Figure 19: In vitro transcription of ∆61 constructs of wild type and derivatives. In vitro transcription assay was performed from supercoiled (σ = -0.075) and linearized templates. Transcripts originating from bla gene on the same plasmid is also shown. SC – supercoiled templates; lin – linear templates. The tyrT wild-type and tyrTD promoter constructs, which clearly showed sensitivity to supercoiling, were investigated in more detail to elucidate the extent of activation by FIS at different superhelical densities. The activity of the wild-type promoter showed a more strict dependence on supercoiling level for its activity, while tyrTD was active under a broader range of superhelical densities (Figure 20A). Presence of FIS was seen to activate the transcription at all supercoiling levels in both wild-type and the discriminator mutant, but the activation of the wild-type was more significant under non-optimal supercoiling levels (σ = - 0.036 & -0.068) (Figure 20B). 3.2.5 Effects of promoter mutations on the function Since the data obtained implicated the mutations in the core promoter region in the altered sensitivity to both supercoiling and FIS we next investigated the effects of these mutations on promoter function by using potassium permanganate footprinting 54
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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
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 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: RESULTS parC) which allows the supe
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
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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