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

More documents

Recommendations

Info

RESULTS 3.2 Role of supercoiling and chromatin protein FIS in transcriptional regulation of an individual promoter The current section describes the role of supercoiling and FIS in activation of the promoter of one of the stable RNA promoters, tyrT. The obtained results give an insight into the mechanistic basis of interplay of these regulators at the molecular level. 3.2.1 Mechanism of transcriptional regulation by FIS & supercoiling: tyrT promoter paradigm It is known that the tyrT and other stable RNA promoters are regulated by FIS and negative supercoiling [Lazarus & Travers, 1993; Muskhelishvili et al., 1995 and 1997; Free & Dorman, 1994; Bowater et al., 1994]. Figure 16A shows the sequence organization of tyrT promoter. To address the role of the core promoter structure in the response of the tyrT promoter to activation by FIS and regulation by supercoiling, three different “up” mutations were introduced in the tyrT core promoter region: a -35 “up” mutation (35U), a discriminator mutation (D), and an A-T base-pair insertion at position -22 in the spacer region between the -10 and -35 elements (S) (Figure 16B). The mutations in the -35 region and in the spacer (35U and S) increase the homology of the promoter to the consensus polymerase binding sequence and the discriminator mutation (D) relieves the block imposed by the G/C-rich discriminator. Both the wild-type and truncated promoter constructs lacking sequences beyond position -61 and thus devoid of UAS with all three FIS binding sites were used for the study. These latter promoter constructs are designated ∆61 promoters. 49
RESULTS Figure 16: Schematic representation of tyrT promoter. (A) Shows the position of three upstream FIS binding sites (I, II & III) and the other core promoter elements. (B) The Sequences of core regions of tyrT and its derivatives are shown. The spacer region and the – 35 and – 10 and discriminator elements are indicated. The substitutions in the discriminator and – 35 hexamer regions and the insertion in the spacer region are highlighted in bold. The consensus – 35 and – 10 elements are shown for reference below. 3.2.2 In vivo expression of tyrT in CSH50 Wt & fis mutant The in vivo activity of tyrT promoter was investigated by transforming the tyrT promoter – lacZ fusion construct ptyrT (Wt) and its derivatives ptyrTD (D), ptyrTS (S), ptyrT35U (35U) into CSH50 wild-type and CSH50 fis::kan cells. The corresponding UAS truncated versions (∆61 constructs) of all the core promoter mutants and the wildtype were also investigated to distinguish the effects of core promoter from those of FIS and the UAS region. Figure 17 shows the averaged β-Galactosidase activities from two independent experiments normalized to the activity of full length wild-type promoter set at 100%. The higher activities of the mutant promoters compared to both the full length and ∆61 wild-type constructs can be explained by the introduced up mutations. The observed absence of difference between wild-type and fis cells in the case of full length promoters is consistent with previous finding which suggests a de-repression mechanism of stable RNA promoters operating in fis cells [Lazarus & Travers, 1993]. 50
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 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: RESULTS Table 4: RT-PCR analysis of
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
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 117 and 118:
APPENDIX I gene blattner ratio p-va
Page 119 and 120:
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
APPENDIX I Supplementary table - VI
Page 129 and 130:
APPENDIX I fabB b2323 0.6336 0.0043
Page 131 and 132:
APPENDIX I ibpA b3687 2.4960 0.0181
Page 133 and 134:
APPENDIX I pepP b2908 1.2182 0.0368
Page 135 and 136:
APPENDIX I speG b1584 0.4151 0.0001
Page 137 and 138:
APPENDIX I ycjX b1321 4.0841 0.0141
Page 139 and 140:
APPENDIX I yhfY b3382 1.4063 0.0453
Page 141 and 142:
Page 143 and 144:
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151 and 152:
Page 153 and 154:
Page 155 and 156:
Page 157 and 158:
APPENDIX I gene blattner ratio A/B
Page 159 and 160:
Page 161 and 162:
Page 163 and 164:
Page 165 and 166:
Page 167 and 168:
Page 169 and 170:
Page 171 and 172:
Page 173 and 174:
Page 175 and 176:
Page 177 and 178:
Page 179 and 180:
Page 181 and 182:
Page 183 and 184:
Page 185 and 186:
Page 187 and 188:
Page 189 and 190:
Page 191 and 192:
Page 193 and 194:
Page 195 and 196:
Page 197 and 198:
Page 199 and 200:
Page 201 and 202:
Page 203 and 204:
Page 205 and 206:
Statement of sources Declaration I
Page 207:
190
show all

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

Create successful ePaper yourself

Delete template?

Save as template?