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

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INTRODUCTION Unwinding of the -10 hexamer and downstream sequences towards the start point is strongly facilitated by negative supercoiling. Furthermore, the necessity for supercoiling is seen in the geometry of arrangement of -10 & -35 hexamer sequences, the principal recognition elements for RNAP holoenzyme. The consensus spacer length of 17bp aligns the two hexamer sequences in an optimal orientation for RNAP binding. Variations in the spacer length would require changes of twist (Figure 5) for optimal alignment of the –10 and –35 elements. Figure 5: Schematic representation of core promoters with different spacer length. 1.2.3 DNA Supercoiling and mechanisms regulating it The DNA inside the cell adopts many different structural forms to suit differenet processes like transcription, replication, recombination etc. All the different processes pose a challenge to maintain the lengthy polymer (DNA) intact and compact. Since the movements of individual nucleotides in the polymer have only certain degrees of freedom, the compensatory adjustments to relieve the helical tension are realized by coiling of one double helix over the other, at which the DNA is said to be ‘supercoiled’. The supercoiling can either be positive or negative. The supercoiling is said to be negative when the DNA strands are under-wound, that is, each 360 o twist of DNA will 9
INTRODUCTION have more base-pairs than there would be in a relaxed molecule of DNA (10.4 bp/turn). The superhelical tension can be relieved by nicking one of the strands of DNA, rotating and re-ligating the strands. The other option would be to convert the helical tension of the twist into writhe, by which the DNA attains the supercoiled form. The writhe in the case of negative supercoiling will be right-handed. The DNA is said to be positively supercoiled when there are fewer base-pairs per helical turn compared to a relaxed molecule. The superhelical tension of twist in this case can be relieved by introducing left-handed writhe. The supercoiling of the DNA can be described using three parameters: [1] Linking number (Lk) – an integer value which refers to the number of times the two strands make a 360 o turn [2] Twist (Tw) – a real number denoting the winding of strands around the helical axis [3] Writhe (Wr) – a real number which denotes the supercoils in the DNA. The three parameters are related as follows: Linking number = Twist + Writhe (Lk = Tw + Wr). As said before, most DNA in vivo is negatively supercoiled. The relative degree to which a molecule is supercoiled can be expressed as the specific linking difference or superhelical density (σ). This quantity gives a lengthindependent measure of the level of supercoiling, σ = (Lk - Lk o )/Lk o . According to ‘twin supercoiled domain’ model, transcription generates positive supercoils in front of the transcribing RNAP complex and negative supercoiling behind it [Liu and Wang, 1987; Wu et al., 1988]. Topoisomerases are the enzymes which take care of these two domains of superhelical tension. There are four topoisomerases in E.coli. Topoisomerase II (gyrase, product of gyrA and gyrB genes) introduces negative supercoils while the other three topoisomerases, topoisomerase I (topA gene product), III (topB gene product) and IV (parC and parE gene product) relax negative supercoils. Gyrase, topoisomerase I & IV are held responsible for maintaining the supercoiled state of the DNA while topoisomerases III mainly manages decatenation reactions [Hiasa and Marians, 1994]. The negatively supercoiled DNA inside the cell is maintained at a steady state level by the actions of gyrase, topoisomerases I and topoisomerase IV [ Menzel et al., 1983; Zechiedrich et al., 2000] within + 15% range of normal mean value [Drlica et al., 1992]. The feedback mechanisms involved in the expression of gyrA, gyrB (active at 10
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: INTRODUCTION can also influence elo
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 and 74: RESULTS contrast the tyrTD promoter
Page 75 and 76: 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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