complementation of Rts1 RepA with phage p1 RepA protein

Rts1 RepA 与噬菌体 p1 RepA 蛋白的互补

基本信息

批准号：
05454191
负责人：
TERAWAKI Yoshiro
金额：
$ 4.1万
依托单位：
School of Medicine, 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：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454191/
关键词：
Plasmid Rts1 Phage P1 Initiator protein RepA Hybrid protein ori activation Autorepression DNA binding Replication inhibition オートリプレッションキメラ蛋白質機能的ドメイン DNA結合能不和合性

项目摘要

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of replication. To study the functional domains of RepA,hybrid proteins were constructed of Rts1 RepA with the RepA initiator protein of plasmid P1 such that the N-terminal portion was from Rts1 RepA and the C-terminal portion from P1 RepA.Six hybrid protein were examined for function.The minimal region of Rts1 RepA required for the in trans activation of Rts1 origin encompassed the N-terminal 206 amino acids while only 129 N-terminal amino acids were required for binding to the Rts1 ori region in vitro. Both in vivo and in vitro studies showed that the N-terminal 257 amino acids fragment was required for autorepressor activity. These results cleary indicate that the N-terminal region of the RepA molecule of Rts1 is involved in the activation of the replication origin of Rts1.Among the hybrid proteins, RepAX15 consisting of the N-terminal 145 amino acids of Rts1 RepA and 142 amino acids from P1 RepA is most interesting, since it showed strong interference with both Rts1 and P1 replication, although the protein could not bind in vitro to P1 ori efficiently. It should be also stressed that no hybrid RepA activated P1 ori. These results suggest that the C-terminal region of both Rts1 and P1 RepA is involved in protein-protein interaction, perhaps in dimer formation.

由288个氨基酸组成的质粒RTS1的REPA蛋白是一种用于启动复制必不可少的跨作用蛋白。为了研究REPA的功能结构域，与质粒P1的REPA引发剂蛋白构建了混合蛋白，使N端部分来自RTS1 REPA，并检查了P1 Repa.six Hybrid蛋白的C端部分。功能。RTS1原点的反式激活所需的RTS1 REPA的最小区域包含N末端206氨基酸，而仅需要129个N末端氨基酸才能在体外与RTS1 ORI区域结合。体内和体外研究都表明，自动压力活性需要N末端257氨基酸片段。这些结果清晰表明，RTS1的RepA分子的N末端区域参与RTS1的复制起源的激活，杂交蛋白，由RTS1 REPA和142氨基酸的N末端145氨基酸组成的混合蛋白，Repax15从P1 RepA中最有趣，因为它表现出对RTS1和P1复制的强烈干扰，尽管该蛋白质无法在体外有效与P1 ORI结合。还应强调，没有混合repa激活的P1 ORI。这些结果表明，RTS1和P1 REPA的C末端区域均参与蛋白质 - 蛋白质相互作用，也许是在二聚体形成中。