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

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DISCUSSION fis experiment show both activation and repression by FIS especially under conditions of relaxation. Whilst, as expected the effect of hns mutation was most pronounced for the Rep genes, these Rep genes predominated the entire growth cycle and were equally enriched under both hypernegative and relaxed conditions. In summary variable distribution of supercoiling sensitivity of transcripts as seen from the genomic wheels (Figure 13) reflect a compound effect of several cooperating factors, including chromatin proteins FIS and H-NS. The genomic wheel picture of transcript profile gives an idea of (1) the supercoiling distribution during different growth phases throughout the genome (2) the effect of different chromatin proteins on such distribution, and (3) also can help in defining the domain boundaries, formation of which is probably a result of differential supercoiling sensitivity or differential NAPs binding in the genome. The data so far supports the idea that transcription is governed by “effective” superhelicity – a parameter reflecting the dynamic nature of competition between topoisomerases, NAPs and also the transcription machinery itself [Travers & Muskhelishvili, 2005b; Geertz et al., 2007]. 4.2 Supercoiling coordinates metabolic functions The changes of supercoiling levels inside the cell not only affect global genome structure and the formation of supercoiling domains with the help of NAPs, but also mediate the change of metabolic preferences. Supercoiling is known to act as a sensor of external environment, whereby the supercoiling level changes in response to nutrition, stress, temperature and ionic content [Balke & Gralla, 1987; Cheung et al., 2003; Dorman, 1996; Travers & Muskhelishvili, 2005a]. It is feasible to imagine the bacterial cell accumulating the energy during exponential growth when nutrition is freely available and storing it in the form of increased torsional tension in the DNA. Put other way, an increase in nutrition leading to increase in ATP/ADP ratio will increase gyrase activity making the DNA more negatively supercoiled. Also increased cell division during exponential growth will have to match with increased requirement for biosynthesis functions. During unfavourable conditions when the nutrition is sparse the 61
DISCUSSION cells maintain viability by deriving energy from degradation of macromolecules. The current work provides evidence for the first time of a relation between the genes involved in biosynthetic and degradation pathways and supercoiling preference of gene activities. Table 2 illustrates that the biosynthetic pathways are made up predominantly of Hyp genes while the degradation pathways prefer Rel genes, implicating the supercoiling as global regulator of metabolic functions. A more detailed analysis of the gene promoters with respect to their sequence organization, their topological properties, their preference of sigma factors and any NAP binding can help to better understand the mechanistic basis of the functional switch from biosynthesis to degradation. The tricaboxylic acid (TCA) cycle and the glyoxylate bypass provide an example for coupling of supercoiling sensitivity to metabolic function. The TCA cycle (Figure 15A) plays two essential roles in metabolism. First, the cycle is responsible for the total oxidation of acetyl coenzyme A (CoA), derived mainly from the pyruvate produced by glycolysis. Second, TCA cycle intermediates are required in the biosynthesis of several amino acids. For long the TCA cycle was considered as housekeeping pathway, but of lately evidences show that it is an inducible pathway [Iuchi & lin, 1988; Iuchi et al., 1989; Guest & Russell, 1992]. The full TCA cycle in E.coli is seen only during aerobic growth on acetate or fatty acid and under anaerobic conditions the cycle is not used for energy production but used more as a biosynthetic pathway. The growth on acetate or fatty acids also results in the induction of the glyoxylate shunt, which is the biosynthetic pathway to replenish the intermediates for amino acid biosynthesis. The gene icdA which codes for IDH (isocitrate dehydogenase) plays a key role in partition of carbon between TCA cycle and glyoxylate shunt. The switch is controlled by phosphorylation of IDH which inactivates IDH and activates the glyoxylate bypass. The genes in the bypass show preference for hypernegative supercoiling while the suc and sdh genes of the TCA cycle are more active under relaxation of DNA. Thus hypernegative supercoiling reflecting increased gyrase activity resulting from a high ATP/ADP ratio favours the glyoxylate bypass and substrate synthesis. Activation of the suc and sdh gene operons under conditions of DNA relaxation (low ATP/ADP ratio) helps in capturing the energy released by carbon metabolism. The switching gene icdA itself is also sensitive to hypernegative 62
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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
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INTRODUCTION Salmonella: “In more
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INTRODUCTION Figure 1: Schematic il
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INTRODUCTION UP element -35 -10 Spa
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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 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: DISCUSSION In a study by Balke & Gr
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
Page 127 and 128: 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 p-va
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APPENDIX I gene blattner ratio A/B
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Statement of sources Declaration I
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