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Structure and Function of the RodA Protein Responsible for the Rod Shape of Escherichia coli

负责大肠杆菌杆状形状的 RodA 蛋白的结构和功能

基本信息

批准号：
62560072
负责人：
MATSUZAWA Hiroshi
金额：
$ 1.34万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62560072/
关键词：
Escherichia coli rod shape of bacteria cell division peptidoglycan synthetase the rodA gene RodA protein transmembrane structure of a protein 遺伝子工学融合タンパク質

项目摘要

In this study, the structure and function of the RodA protein of Escherichia coli were analysezed by the methods of protein chemistry and gene engineering.1. the rodA gene is located 5 nucleotides downstream of the pbpA gene, encoding penicillin-binding protein (PBP) 2. Overproduced RodA protein was purified by SDS-PAGE. The determined N-terminal amino acid sequence showed that the protein has no cleavable signal sequene, but its N-terminal Met residue is removed.2. To determine the transmembrane structure of the RodA protein, plasmids carrying rodA-bla gene fusions were constructed. Based on the ampicillin resistance of the cells producing RodA-beta-lactamase fusion protein and the hydropathy of the RodA protein at the fused region, localization of the fused point was determined to be in the cytoplasm, cytoplasmic membrane or periplasm. The results obtained showed that the RodA protein traverses the membrane eight times with its N- and C-terminals in the cytoplasm.3. The ftsL gene is the structural gene for PBP 3. sui mutation was reported, which is a kind of rodA mutation and suppresses a temperature-sensitive lethal mutation of ftsL. The roda(sui) gene was cloned, and its determined nucleotide sequence indicated that the sui mutation is a mutation of CAG (Gln-III) to amber termination codon, TAG, in the rodA gene. Suppressor strain KJBI, which contains both ftsL(Ts) and sui mutations, also has supE mutaion. supE can partially replace the termination signal with Glh. It is considered that the reduction of the RodA protein caused by sui mutaion suppresses the low activity of PBP 3. It is suggested that the RodA protein, probably as a RodA-PBP 2 complex, is involved in the cell division together with PBP 3.

本研究采用蛋白质化学和基因工程的方法对大肠杆菌RodA蛋白的结构和功能进行了分析。rodA基因位于pbpA基因下游5个核苷酸处，编码青霉素结合蛋白（PBP） 2。过量生成的RodA蛋白经SDS-PAGE纯化。测定的n端氨基酸序列表明该蛋白没有可切割的信号序列，但其n端Met残基被去除。为了确定RodA蛋白的跨膜结构，构建了携带RodA -bla基因融合物的质粒。根据产生RodA- β -内酰胺酶融合蛋白的细胞对氨苄西林的耐药性和融合区RodA蛋白的亲水性，确定融合点的定位在细胞质、细胞质膜或外周质。结果表明，RodA蛋白的N-和c -末端在细胞质中穿过细胞膜8次。ftsL基因是PBP - 3的结构基因。sui突变是一种抑制ftsL温度敏感致死突变的rodA突变。克隆了roda（sui）基因，测定其核苷酸序列表明，sui突变为roda基因中CAG （Gln-III）向琥珀色终止密码子TAG的突变。抑制菌株KJBI同时存在ftsL（Ts）和sui突变，也存在超e突变。supE可以用Glh部分代替终止信号。认为sui突变引起的RodA蛋白的减少抑制了PBP 3的低活性。提示RodA蛋白可能以RodA-PBP - 2复合物的形式与PBP - 3共同参与细胞分裂。