| Project/Area Number |
11480200
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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 | KANAZAWAI UNIVERSITY |
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Principal Investigator |
MURAKAMI Seishi KANAZAWAI UNIVERSITY, Dept. Mol. Oncology, Cancer Res. Inst., Professor, がん研究所, 教授 (90019878)
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| Co-Investigator(Kenkyū-buntansha) |
HAYASHI Naoyuki KANAZAWAI UNIVERSITY, Dept. Mol. Oncology, Cancer Res. Inst., Research Associat, がん研究所, 助手 (50253456)
NOMURA Takahiro KANAZAWAI UNIVERSITY, Dept. Mol. Oncology, Cancer Res. Inst., Assistant Professor, がん研究所, 助教授 (80115261)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥11,000,000 (Direct Cost: ¥11,000,000)
Fiscal Year 2001: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1999: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Keywords | RNA polymerase II subudit 5(RPB5) / Transcriptional regulation / Hepatitis B Virus X protein / RMP / TFIIF / Transcriptional cofactors / RNAポリメラーゼサブユニット5(RPB5) / 転写制御 / HBVX蛋白 / RNAポリメラーゼ / RPB5 / RAP30 / 転写修飾 / B型肝炎ウイルス / X蛋白 / TFIIB |
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| Research Abstract |
RPB5 at the tip of the lower jaw of pol II is involved in activated transcription and interacts with basal transcription factors and Hepatitis B Virus X protein (HBx). A crystal model of pol II predicted RPB5 is close to DNA downstream to transcription start site. 1) RPB5 was found to bind to recombinant TFIIF reconstituted consisting of RAP30 and RAP74. The central parts of RAP30 and RPB5 are critical for the binding. By scanning clustered, then point alanine-substitution mutants of RAP30, two residues, Y124 and Q131, were found to be critical for the RPB5-binding. Endogenous pol II was recruited to TFIIF consisting of wild RAP30 but not mutant RAP30 at aa124 or at aa131. 2) We found that RPB5 retains ability to bind to double-stranded DNA. The 4 amino acid residues in the exposed domain of RPB5 were identified to be critical for the DNA- binding analyzed by scanning alanine substitution mutants of the exposed domain. Among them, T111 and SI13 would be directly involved in the DNA-binding. 3) RMP (RPB5-mediating protein), a functional antagonist of HBx, has a coiled-coil domain at the N-terminal, RPB5-binding region, and the C-terminal region responsible for co- repressor activity. The coiled coil domain harboring cytoplasmic localization signal (CLS) and a NLS located at the C-terminal region are both important for cytoplasmic and nuclear localization of RMP. 4) To analyze function(s) of RMP, RMP-interacting partners were searched by yeast two hybrid screening. RMP, by itself, was screened and found to interacts with RMP in vitro and in mammalian cells. Another candidate, repeatedly screened out, has been reported to be a corepressor and involved in DNA methylation. Biological outcome of the interactions remains to be examined.
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