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

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INTRODUCTION Furthermore, expression of a σ factor (σ 70 ) can in turn affect the global topology of DNA [Geertz et al., 2007]. Figure 8: Growth phase-coupled change in the level of different forms of the RNA polymerase. (RpoD: sigma70; RpoN: sigma54; RpoS: sigma35; RpoH: sigma32 ; RpoF: sigma28; RpoE: sigma24; FecI: sigma19)[Ishihama et al., 1999]. 1.3 Organization of global transcription - coordinated view The previous sections addressed the growth dependent alterations in factors having a global influence including DNA superhelical density, abundant DNA binding proteins and the transcription machinery. The present section describes interactions between these global regulators under variety of conditions where they exert their control. The E.coli genome compacted ~1000 times inside the cell manages to coordinate gene expression with growth phase by forming spatially differentiated domains. Domains, defined as topological barriers preventing diffusion of supercoiling, are dynamic entities. Reported domain sizes vary from 10 to 100 kb (kilo base-pairs) per domain [Postow et al., 2004; Sinden & Pettijohn, 1981]. The topological barriers 15
INTRODUCTION formed as a repercussion of active transcription from highly transcribed gene by removing any previously established DNA attachments would generate barriers near the transcription terminus [Deng et al., 2005]. Barrier formation is also altered by abnormal activities of gyrase and topoisomerase IV enzymes [Staczek & Higgins, 1998]. Scattered binding sites for NAPs in the genome may also act as regions where proteins bind to prevent the diffusion of supercoiling. DNA supercoiling itself might act as a principal coordinator of global gene expression [Balke & Gralla, 1987; Dorman, 1996; Tse-Dinh et al., 1997; Cheung et al., 2003; Travers & Muskhelishvili, 2005b]. Recent enumeration of number of supercoiling sensitive genes in the E.coli genome found only 8% of the total genes to be associated with supercoiling [Peter et al., 2004]. However the observed role of supercoiling in many distinct processes such as sigma factor recognition, transcription termination, sensing of stress and nutritional status appeal to a much wider influence of supercoiling. Furthermore, recent study of the experimental evolution of E.coli populations identified supercoiling as a key target for evolution and mutations in topA & fis genes were found to conferred increased fitness [Crozat et al., 2005]. The supercoiling transitions during growth are tightly coupled to the changing NAP composition and abundance. During early exponential phase the nucleoid is composed of FIS, HUα2 and H-NS proteins, to be succeeded by HUαβ and H-NS in the transition between exponential and stationary phase, and finally by IHF and Dps in late stationary phase. Among these FIS, HU, HNS constrain different amounts of supercoiling [Travers & Muskhelishvili, 2005a], resulting in a change of plasmid DNA supercoiling when the constraining proteins are eliminated [Schneider et al., 1997; Berger M et al., unpublished; Blot et al., 2006]. FIS binds to the DNA as a dimer and can bend DNA by 50-90 o [Pan et al., 1996]. Low amount of FIS binding has a distinct effect on the architecture of DNA compared to multiple FIS binding. Low amounts of FIS lead to the formation of branches on the supercoiled DNA, while binding in large amounts it compacts the DNA [Schneider et al., 2001]. HU present in comparable amounts to FIS binds non-specifically to supercoiled DNA and to structurally distorted DNA [Pontiggia et al., 1993]. H-NS present in 20,000 copies binds patches of DNA with a preference for A/T rich tracts and functions as a general repressor of transcription 16
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: INTRODUCTION shown that H-NS reache
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 and 80: DISCUSSION cells maintain viability
Page 81 and 82: DISCUSSION writhe in the UAS by FIS
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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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