Analysis of the DNA binding and replication initiation capabilities of RepA protein

RepA蛋白的DNA结合和复制起始能力分析

• 批准号: 02454177
• 负责人: TERAWAKI Yoshiro
• 金额: $ 3.33万
• 依托单位: Shinshu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454177/
• 关键词: 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

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.

由288个氨基酸组成的RTS1的REPA蛋白是启动RTS1-DNA复制必不可少的反式作用蛋白。但是，低频构型构型。 °C。我们构建了另一个编码REPA的杂种REPA基因（RTS1：P1）-2，该基因由RTS1-REPA的N端114氨基酸和P1-REPA的174 C-末端氨基酸组成。 RTS1）即使当杂交CENE与ORI的自然构型处于自然构型中（RTS1）。从N端到AA114的N末端多肽。使用PFD51作为Areporter质粒，通过G ALK表达系统检查了REPA的自动压力。 PFGY1，使自动压活动成为可能。

项目成果

曽 虹: "野性型及び変異RepA蛋白質のin vivo安定性,ならびにRepA蛋白質N末端の機能解析" 日本細菌学雑誌. 46. 442 (1991)

曾宏：“野生型和突变型 RepA 蛋白的体内稳定性以及 RepA 蛋白 N 末端的功能分析”日本细菌学杂志 46. 442 (1991)。

Y.Terawaki: "Function of the Nーterminal half of RepA in activating ori(Rts1)" J.Bacteriol.

Y.Terawaki：“RepA 的 N 末端一半在激活 ori(Rts1) 中的功能”J.Bacteriol。

Zeng,H.: "Site-directed mutations in the repA C-terminal region of plasmid Rts1:pleiotropic effects on the replication and autorepressor functions." J.Bacteriol.172. 2535-2540 (1990)

Zeng,H.：“质粒 Rts1 的 repA C 端区域的定点突变：对复制和自抑制功能的多效性影响。”

Terawake,Y.: "Effects of mutations in the repA gene of plasmid Rtsl on plasmid replication and autorepressor function." J.Bacteriol. 172. 786-792 (1990)

Terawake,Y.：“质粒 Rtsl 的 repA 基因突变对质粒复制和自抑制功能的影响。”

寺脇 良郎: "Rts1複製必須蛋白質RepAの機能解析" 日本細菌学雑誌. 47. 196 (1992)

Yoshiro Terawaki：“Rts1 复制必需蛋白 RepA 的功能分析”《日本细菌学杂志》47. 196 (1992)。