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

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INTRODUCTION 1 Introduction Cellular division, differentiation and development implicate gene expression programs, which are coordinated in time and space both in prokaryotes and eukaryotes. In prokaryotes, control of transcriptional initiation is the most pervasive form of gene regulation whereby recent development of high-throughput technologies allow to analyze simultaneously the activities of thousands of genes. Prokaryotes like E.coli K12 (non-pathogenic laboratory strain) give us in addition a possibility to study genetic regulation under wide range of variation in external physical and chemical parameters such as temperature, pH, barometric pressure, osmolarity, oxygen availability, nutrient supply and presence of toxic substances. Furthermore in this classical model organism a large amount of genetic information has already been accumulated, providing a unique opportunity for genome-wide analyses of global gene expression patterns. E.coli is a facultative anaerobic, gram-negative bacterium growing at rates varying from 20 minutes to 24 hours depending on the environment. This organism thus shows an impressive ability to grow and divide rapidly but can grow slowly for prolonged times as well. E.coli growing in rich medium reproduces fast and does so primarily by elevating the expression levels of genes for ribosomal proteins and stable RNA, and other translation machinery factors. In contrast, in minimal medium expression levels of genes involved in biosynthesis of building blocks, notably amino acid biosynthesis are elevated [Wei et al., 2001; Tao et al., 1999; Weber et al., 2005]. Both these conditions are also associated with numerous subtle changes like redirection of intermediary metabolism, changes in electron transport chain composition and concentration of different metabolic enzymes [Murray et al., 1996; Neidhardt et al., 1987). During the growth, also a number of morphological and physiological attributes change. Investigation of gene expression patterns under different growth conditions reveals a distinct arrangement of expressed functions, indicating that E.coli possess proprietary over a multitude of gene expression programs. Heterogeneity of E.coli’s response is exemplified as follows in the classical book Escherichia coli and 1
INTRODUCTION Salmonella: “In more than just a figurative sense, bacteria invented gene regulation, and the bacterial cell’s style of growth uniquely involves second-by-second adjustment in the flow of information from the genome to ribosomes. Individual mRNA molecules function for only a few minutes before being destroyed and replaced by newer, more timely information”. The fundamental problem addressed is not so much the understanding particular gene expression programs but rather the mechanistic basis of the concerted rearrangements of these programs. One incentive of this work therefore is to reveal the mechanism coordinating the growth phase-dependent transcription in E.coli. for this we have developed a holistic approach, using microarrays and a combination of other in vitro and in vivo techniques, to demonstrate how differential spatio-temporal distributions of supercoiling sensitivity in the genome effected by NAPs coordinate growth phase dependent transcription patterns. 1.1 Is there a common coordinating factor? A typical growth cycle of E.coli growing in batch culture when plotted against time describes a sigmoidal curve with three clearly discernible stages. Correlations have been observed between different stages along the sigmoidal curve, called phases of growth, and the global gene expression patterns [Wei et al., 2001; Tao et al., 1999; Weber et al., 2005]. To better understand this correlation it is essential to identify those factors which have global impact on the transcription process. In E.coli the transitions between the different growth phases are associated with remarkable alterations of three parameters that can affect cellular transcription: the protein composition of the nucleoid, the global supercoiling level of DNA and the composition of transcriptional machinery. The two predominant RNA polymerase (RNAP) holoenzyme forms (based on number of genes they are known to transcribe) operating in E.coli, Eσ 70 and Eσ s (RNAP holoenzyme associated with sigma subunit 70 and sigma s) differ in their DNA topology requirements. The topological state of cellular promoters depends on the partitioning of unconstrained (i.e. free) and constrained (i.e. fixed by interactions with 2
Page 1 and 2: Coordinated regulation of gene expr
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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: ABBREVIATIONS TBE Tris TS tyrT35U t
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
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Page 31 and 32: INTRODUCTION shown that H-NS reache
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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
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Page 45 and 46: MATERIALS AND METHODS phenol: chlor
Page 47 and 48: MATERIALS AND METHODS Filtered (by
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Page 51 and 52: RESULTS 3 Results Growth phase-depe
Page 53 and 54: RESULTS Neither the fis nor the hns
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Page 57 and 58: RESULTS furthermore the colour of t
Page 59 and 60: RESULTS During the entire growth cy
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RESULTS parC) which allows the supe
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RESULTS Figure 19: In vitro transcr
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RESULTS contrast the tyrTD promoter
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DISCUSSION 4 Discussion The idea of
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DISCUSSION In a study by Balke & Gr
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DISCUSSION cells maintain viability
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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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