| Project/Area Number |
02454552
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
分子遺伝学・分子生理学
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| Research Institution | Kyoto University |
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Principal Investigator |
SHIGESADA Katsuya Kyoto University, Institute for Virus Research, Associate Professor, ウィルス研究所, 助教授 (40009626)
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| Co-Investigator(Kenkyū-buntansha) |
TSURUSHITA Naoya Kyoto University, Institute for Virus Research, Instructor, ウィルス研究所, 助手 (30201643)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1991: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Transcription termination / Rho factor / Escherichia coli / PCR-mediated mutagenesis / Expression vector / Terminator / RNA binding / RNA-dependent ATPase / PRC変異誘発法 / 転写終結因子 / 大腸菌転写制御 / PCR / in vitro 変異誘発 |
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| Research Abstract |
The Escherichia coli transcription termination factor rho is a hexamer of 46kDa subunits and catalyzes the release of the nascent RNA from the ternary transcription complex. A rho monomer is known to have three major structural domains : the N-terminal one third possessing an RNA binding activity, the central 250-aa region carrying the ATP binding site and the C-terminal remainder of an unknown function (s). To further elucidate the precise structure-function relationships of rho protein, we introduced random mutations into its individual structural domains using a modified PCR reaction. The mutagenized rho alleles were cloned into a lac promoter-controlled expression vector , pEC22, and selected for their impaired rho-dependent termination activities. We first focused on the least-characterized C-terminal domain, and isolated 53 clones carrying point mutations within the 3'-terminal 310nt region of the rho-coding sequence. Thus far, rho proteins from two such mutants, E342G and A357V, were purified and characterized in vitro. The results revealed that the both mutations affect the mode of recognition of RNA and the efficiency of ATP hydrolysis by rho protein Thus the C-terminal domain must have a vital role in regulating and coordinating the functions of the RNA binding and ATP binding domains.
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