| Project/Area Number |
04454538
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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 |
Human genetics
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
YASUKOCHI Yukio Tokyo Medical and Dental University Medical Reaearch Institute Professor, 難治疾患研究所, 教授 (60037398)
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| Co-Investigator(Kenkyū-buntansha) |
CHIBAZAKURA Taku Tokyo Medical and Dental University Medical Reaearch Institute Reseach Associate, 難治疾患研究所, 助手 (30227334)
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| Project Period (FY) |
1992 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥7,300,000 (Direct Cost: ¥7,300,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1993: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1992: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Keywords | TFIIF / phosphorylation / DBPolF complex / DEPolF複合体 / 染色体マッピング / RAP30 / 74 / DBPolF複合体 / バクテリア性RNA polymerase / HeLa細胞 / 一般転写因子 |
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| Research Abstract |
The strucural and functionl domains of a general transcription initiation factor, TFIIF (RAP30/74, FC) , have been investigated using various deletion mutants of each subunit, both in vivo and in vitro. An in vivo assay showed that the N-terminal sequence containing resudues of 1-110 of RAP30 that is located close to a sigma homology region interacts with a minimum sequence of residues 62-171 of RAP74 to form a heteromeric interaction. Reconstitution of in vitro transcription activity by deletion mutants of RAP74 clearly indicated that both N-terminal residues 73-205 and C-terminal residues 356-517 are essential for full activity, the former interacting with RAP30, thus complexing with RNA polymerase II.Furthermore in the course of research we noticed the posttranslational modification of TFIIF.The possible regulation of TFIIF activity by phosphorylation was investigated by comparing the biochemical properties of alkaline phosphatase-treated HeLa TFIIF with those of native or bacterially expressed factor. Alkaline phosphatase treatment decreased the size of the larg subunit (RAP74) of TFIIF to that of the recombinant protein but did not change the size of the small subunit (RAP30). Both the transcription initiation and elongation stimulating activities of the alkaline phsophatase-treated TFIIF decreased to 15-20 % of the native form under conditions in which the amount of TFIIF was late-limiting for transcription.Furthermore phosphatase-treated TFIIF assembled the DBPolF complex and bound to RNA polymerase less efficiently than the native protein. When hybrid TFIIFs were reconstituted using native or recombinant subunits, a native form of RAP74 stimulated both transcription and DBPolF complex formation activity regardless of whether native or recombinant RAP30 was used. We propose that TFIIF activity is regulated by protein phosphorylation, particularly of RAP74 subunit.
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