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

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INTRODUCTION characteristics, the amount of variability in response that can be brought about by factors like NAPs and DNA supercoiling is described, and a brief introduction into how the factors themselves are modulated during growth is outlined. 1.2.1 Gene promoters Transcription initiation is the predominant step in the gene regulation and promoter regions provide binding sites for the different regulatory factors to interact and initiate or repress transcription. The amount of gene product synthesized is primarily determined by the rate of productive initiation. The initiation rates can vary between 1.5X10 3 transcripts per generation to as low as 1 transcript per generation [McClure et al., 1985]. What determines the heterogeneity in the rate of transcription initiation? One of the principal determinants is the promoter sequence itself. Important elements of the promoter sequence are the positions where a change in sequence affects the rate of initiation of transcription from the specified start site. Sequences recognized by Eσ70, the most abundant form of RNA polymerase holoenzyme, are shown in Figure 2. There are at least three features within the “core” region of the promoter DNA: two conserved 6-bp (base pair) DNA sequences centered approximately 10 and 33 bp upstream from the start site of transcription (+1), called the “–10” and “–35” hexamers; and the sequence of DNA that separates them, called the “spacer” region, which is most commonly 17 bp in length but can vary from 15 to 21 bp. Figure 2 summarizes the primary structure of the core promoter, specified by convention in the 5’ to 3’ direction as the sequence of the non-transcribed strand, which corresponds to the transcript sequence. When optimally aligned, the sequences of most promoters match at least 7 of the 12 bp in the consensus –35 (TTGACA) and –10 (TATAAT) hexamers [Lisser et al., 1993]. At many promoters, the primary start site (+1; specified by the same convention) is an A and the flanking bases are C(–1) and T(+2). 5
INTRODUCTION UP element -35 -10 Spacer 17bp Discriminator +1 start Core promoter Figure 2: Structure of consensus Eσ 70 promoter showing functionally important regions. It is now generally accepted that promoter structure and geometry is used by bacteria as a key regulatory element [Perez-Martin et al., 1997]. Computational studies on promoter sequences have corroborated the experimental findings that the promoter regions are more curved and less stable than the average genomic DNA. Observation by Ozoline et al., of the positioning of the highly deformable TA dinucleotide approximately every 5.6 bp, also indicate the distinct flexibility of the promoters [Ozoline et al., 1999]. Analysis of curvature and deformability of the promoters based on their predicted sigma (σ) factor preference also demonstrate variability. From Figure 3, it is evident that the σ 54 class promoters are less deformable compared to σ 70 class. Sigma 54 protein is known to be structurally unrelated to σ 70 , and it also utilizes a different pathway to form open complexes during transcription initiation, which in the case of σ54 requires ATP [Gruber et al., 2003]. It is thought, that the core promoter -35 & -10 hexamer sequences, the discriminator sequence and the spacer region also have evolved to posses regulatory potential. Molecular genetic examinations of stable RNA promoters in E.coli have attributed some of the important regulatory events, like stringent regulation and growth-phase regulation to the core promoter elements [Travers et al., 1980; Josaitis et al., 1995; Auner et al., 2003]. 6
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: INTRODUCTION Figure 1: Schematic il
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
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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
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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 59 and 60: RESULTS During the entire growth cy
Page 61 and 62: RESULTS transcripts of a growth pha
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Page 65 and 66: RESULTS Table 4: RT-PCR analysis of
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Page 71 and 72: 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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