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Regulatory mechnism of cell growth in SOS response of Escherichia coli

大肠杆菌SOS反应中细胞生长的调控机制

基本信息

批准号：
06680672
负责人：
HIGASHITANI Atsushi
金额：
$ 1.47万
依托单位：
National Institute of Genetics
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06680672/
关键词：
SOS response Cell division Cell cycle Inhibitor Protease DNA repair Stress response Single-stranded DNA

项目摘要

Regulatory mechnisms of cell growth in the SOS response of E.coli were investigated with emphas is on SulA protein, an SOS-inducible cell-division inhibitor. Direct interaction of SulA with FtsZ,which plays a central role in bacterial cell division was studied. Molecular dissection of SulA and the role of single-stranded DNA as a primary signal for SOS induction in vivo were also analyzed.1.Using purified SulA protein that was fused to the maltose binding protein, I demonstrated in vitro that SulA interacts with FtsZ to from a stable complex. The reaction required GTP and its hydrolysis. The complex was formed in a molar ratio of approximately one to one of the two proteins.2.From mutation analyzes, Arg 62, Leu 67, Trp 77, and Lys 87 in the central region of SulA were found essential for the cell-division inhibitory activity. N-terminal residues of SulA ranging from the 3rd to the 27th amino acid and C-terminal 21 residues were dispensable for the activity. The C-terminal 20 residues of SulA were enough for its recognition by and for complexformation with Lon protease. They were necessary, but noy enough, for degradation of SulA by Lon protease.3.Infection of E.coli with mutant filamentous phage that are defective in the initiation of minus-strand DNA snythesis induced the SOS response as monitored by cellular level of LexA.This observation demonstrated that single-stranded DNA serves as a primary signal for SOS induction in vivo.

本研究以SOS诱导的细胞分裂抑制剂苏拉蛋白为靶蛋白，研究了SOS反应中大肠杆菌细胞生长的调控机制。研究了苏拉与在细菌细胞分裂中起核心作用的FtsZ的直接相互作用。本研究还对苏拉的分子结构和单链DNA作为SOS诱导信号的作用进行了分析。1.利用纯化的苏拉蛋白与麦芽糖结合蛋白融合，在体外证明了苏拉与FtsZ形成稳定的复合物。该反应需要GTP及其水解。2.突变分析表明，SulA中心区域的Arg 62、Leu 67、Trp 77和Lys 87是SulA细胞分裂抑制活性所必需的。对苏拉的N端第3 ~ 27位氨基酸残基和C端21个氨基酸残基进行了活性测定。苏拉的C端20个残基足以被Lon蛋白酶识别并与其形成复合物。3.用缺失负链DNA合成起始的丝状噬菌体感染大肠杆菌，通过检测细胞内莱克萨的水平，证实单链DNA是体内诱导SOS反应的主要信号。