Analysis of the DNA binding and replication initiation capabilities of RepA protein
| Project/Area Number |
02454177
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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 |
細菌学
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| Research Institution | Shinshu University |
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Principal Investigator |
TERAWAKI Yoshiro Shinshu Univ. School of Med. Dept. Bacteriol. Professor, 医学部, 教授 (10014333)
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| Co-Investigator(Kenkyū-buntansha) |
TABUCHI Akira Shinshu Univ. School of Med. Dept. Bacteriol. Professor, Researcher, 医学部, 助手 (50236725)
NOZUE Hatsumi Shinshu Univ. School of Med. Dept. Bacteriol. Professor, Researcher, 医学部, 助手 (30218325)
HAYASHI Tetsuya Shinshu Univ. School of Med. Dept. Bacteriol. Professor, Researcher, 医学部, 助手 (10173014)
曽 虹 信州大学, 医学部, 助手 (80226696)
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| Project Period (FY) |
1990 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥5,200,000 (Direct Cost: ¥5,200,000)
Fiscal Year 1992: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1991: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1990: ¥4,200,000 (Direct Cost: ¥4,200,000)
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| Keywords | Plasmid Rts1 / Replication / RepA protein / Origin activation / Autorepression / Domain structure / N-terminal function / 複製開始蛋白質 / ori活性化 / ハイブリッド蛋白質 / 機能的ドメイン / プラスミド Rts1 / 複製必須蛋白質 / 複製開始能 / DNA複製 / 変異蛋白質 / 蛋白質の安定性 |
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| Research Abstract |
The RepA protein of the Rts1 plasmid, consisting of 288 amino acids, is a trans-acting protein essential for initiation of Rts1-DNA replication. A mutant repA gene, repADELTAC143, carrying a deletion that removed the 143 C-terminal amino acids of RepA, could transform, but a low frequency, an Escherichia coli polA strain JG112 at 37ﾟC, when repADELTAC143 was cloned into pBR322 with ori(Rts1) in the natural configuration. A fusion of the 3'-terminal half of repA of the P1 plasmid to repADELTAC143 yielded a pBR322 chimeric plasmid that contained ori(Rts1) through hybrid repA(Rts1:P1)-1. This plasmid was maintained much more stably in JG112 at 37ﾟC.
Recently, we constructed another hybrid repA gene encoding RepA (Rts1:P1)-2 that consists of the N-terminal 114 amino acids of Rts1-RepA and the 174 C-terminal amino acids of P1-RepA. RepA(Rts1:P1)-2, however, did not activate ori(Rts1) even when the hybrid repA gene was in the natural configuration with ori(Rts1). These findings suggest that the function to activate ori(Rts1) is located in the N-terminal half of Rts1-RepA spanning from the N-terminus to AA145, but the function is lost when the N-terminal polypeptide was restricted from the N-terminus to AA114.
The autorepressor function of RepA was examined by galK expression system using pFD51 as a reporter plasmid. Recently, we constructed a fusion of the promoter region of Rts1-repA with beta-galactosidase gene of pFGY1, which made possible to express the autorepressor activity quantitatively. By the method, we are analyzing the activity of the hybrid RepA proteins.
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Report
(4 results)
Research Products
(9 results)
