Functional domains of Rts1 RepA analyzed by hybrid proteins with P1 RepA.

通过与 P1 RepA 的杂合蛋白分析 Rts1 RepA 的功能域。

基本信息

批准号：
07457072
负责人：
TERAWAKI Yoshiro
金额：
$ 4.03万
依托单位：
Shinshu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457072/
关键词：
Plasmid Rts1 Phage P1 Initiator protein Domain DNA binding Origin activation Hybrid protein プラスミドRts1 ファージP1 イブリッド蛋白質キメラ蛋白質桟能的ドメイン複製調節プラスミトRts1

项目摘要

1. The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA,hybrid proteins of Rts1 RepA with the prophage P1 RepA,consisting of 286 amino acids, were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA.Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. As an essential subregion of Rts1 RepA for activation of Rts1 ori in vivo, the residues between 177 and 206 was tentatively assigned.2. P1 RepA was found to bind in vitro to Rts1 ori as strongly as Rts1 RepA.In addition, P1 RepA activated in vivo the Rts1 replication origin, although the activation was quite inefficient. In contrast, Rts1 RepA showed neither binding in vitro to P1 ori nor activation in vivo of the origin.3. By replacing a small region of P1 RepA with the corresponding region of Rts1 RepA,the residues 145-176, the efficiency of Rts1 ori activation increased remarkably. The same subregion of P1 RepA was found to be important for in vivo activation of the P1 origin.Thus, a domain essential for an efficient activation of the replication origin was assigned on the P1 RepA molecule as well as on the Rts1 RepA molecule. It should be noted that the domain was distinct from a region necessary for in vitro binding to the origin, although both regions were required for in vivo activation of the replication origin.

1。由288个氨基酸组成的质粒RTS1的RepA蛋白是一种跨作用蛋白，对于启动质粒复制必不可少。为了研究REPA的功能结构域，构造了由286个氨基酸组成的RTS1 REPA的杂化蛋白与预言P1 REPA的构建，以使N末端部分来自RTS1 REPA，C末端部分来自P1 RepA.Six。检查了杂化蛋白的功能。发现氨基酸残基113和129之间RTS1 REPA的N末端区域对于体外的RTS1 ORI结合很重要。作为用于体内RTS1 ORI激活的RTS1 REPA的必不可少的子区域，暂时分配了177至206之间的残基。2。发现P1 REPA在体外结合与RTS1 ORI与RTS1 REPA一样强。加上P1 REPA在体内激活了RTS1复制起源，尽管激活效率很低。相比之下，RTS1 REPA既不在体外与P1 ORI结合，也没有在原始体内激活。3。通过用RTS1 REPA的相应区域（残基145-176）代替P1 REPA的一小部分区域，RTS1 ORI激活的效率显着提高。发现相同的P1 REPA子区域对于P1起源的体内激活很重要。应当指出的是，尽管两个区域在体内激活复制起源所必需的区域都不同于体外结合所必需的区域。