| Project/Area Number |
15370079
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | Nippon Institute for Biological Science |
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Principal Investigator |
ISHIHAMA Akira Nippon Institute for Biological Science, Research Division, Principal Investigator and Lab Head, 研究部, 主任研究員 (80019869)
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| Co-Investigator(Kenkyū-buntansha) |
HONDA Ayae Nippon Institute for Biological Science, Research Division, Senior Scientist, 研究部, 研究員 (80343747)
IWATA Akira Nippon Institute for Biological Science, Research Division, Principal Investigator, 研究部, 主任研究員 (70193745)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥14,400,000 (Direct Cost: ¥14,400,000)
Fiscal Year 2004: ¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 2003: ¥8,200,000 (Direct Cost: ¥8,200,000)
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| Keywords | RNA polymease / Transcript factor / Transcription regulation / Rromoter / Microarray / SELEX / Protein phosphorylation / Transcrption signal / シグマ因子 / 転写調節 / 大腸菌 / タンパク-DNA相互作用 / FeBABE / 二成分制御系 / タンパクリン酸化 |
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| Research Abstract |
The aim of this research was undertaken to understand the molecular basis of transcription regulation of 4,000 genes on the Escherchia coli genome. Previously we found that the total number of RNA polymerase core enzyme in E coli is only 2,000 molecules per genome. The distribution of a limited number of RNA polymerase among 4,000 genes is controlled through modulation of the promoter selectivity of RNA polymerase. For this modulation, 7 species of the sigma subunit (promoter recognition subunit) and about 300 species of transcription factors are involved. In order to reveal the molecular mechanism of RNA polymerase modulation, we have performed several lines of studies as follows :
1)A new promoter assay plasmid vector TFP (two-fluorescent protein) was constructed, into which about 1,000 E. coli promters were inserted. Using this promter eollection, growth phase-coupled variation of the promoter strength has been determined by measuring the expression of GFP/RFP ratio.
2)To identify the
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genes under the control of each sigma or transcription factor, the microarray assay was carried out for E. coli mutants, each lacking one specific factor, using E. coli DNA chip.
3)To identify the DNA sequences recognized by each transcription factor, an improved SELEX method was developed and applied for in vitro screening promoters recognized by each purified transcription factor.
4)The mode of transcription regulation by each transcription factor has been analyzed in vitro using the purified RNA polymerase and transcription factors and the promoters identified by microarray and SELEX.
5)Antibodies against each transcription factor were prepared and used for determination of the intracellular concentration of each sigma and transcription factor under various growth conditions.
6)NIP (nucleoid immuno-precipitation) method was developed to identify the localization in vivo of each transcription factor on the E. coli genome.
7)Kinetics and specificity of regulator phosphorylation by sensor kinase was analyzed for all two-comonent sytem proteins from E. coli. Trans-phosphorylation was observed between cognate pairs but for a limited combinations, the phosphorylation of response regular was observed by non-cognate sensor kinase, indicating the cross-talk in the activation process of transcription factor. Less
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