Study on molecular structure-function relationship of E..coli DnaA protein

大肠杆菌DnaA蛋白分子结构与功能关系的研究

基本信息

批准号：
08680695
负责人：
KATAYAMA Tsutomu
金额：
$ 1.54万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Specific aims : DnaA protein has high affinity for ATP/ADP.Tne ATP-bound form is active for initiation of DNA replication, but the ADP-bound form is not. The first purpose of this work is to reveal mechanism of conformational change of the protein, based on identification of amino acid residue responsible for the ATP-binding. Especially, identification of the residue interacting with phosphate in the gamma position of ATP is most important to know the activation mechanism of the protein. The second purpose is to identify the protein's domain to regulate its activity. For this, dnaA mutants that show lethality for cells bearing the wild-type allele, due to occurrence of excessive initiations will be isolated and biochemical analyzes of the mutant proteins will be performed.Results and Discussion : Affinity labeling using an ATP-analog that contains modified phosphate at the gamma position and determination of amino acid residue that is covalently bound to this analog suggested that Lys-415 residue interacts with the gamma phosphate of ATP.Next, by site-directed mutagenesis this residue was replace with other ones, and such mutant proteins were purified. Analyzes of these suggested that Lys residue is necessary for activation of the protein by ATP (manuscript in preparation). In the second project, by introducing random mutation in the dnaA cistron, we obtained three dnaA mutant genes that show lethality for cells-bearing the wild-type allele. Sequencing indicated that mutations appear in a distinct region between the ATP-binding domain and the DNA-binding domain of DnaA protein. Purification and characterization of a mutant DnaA isolated here revealed that this mutant protein lacks affinity for ATP but sustains replication activity. These results indicate an importance of conformation of this distinct region for regulation of DnaA function, being a critical finding in study of structure function relationship of the protein.

具体目标：DnaA 蛋白对 ATP/ADP 具有高亲和力。ATP 结合形式对于启动 DNA 复制具有活性，但 ADP 结合形式则不然。这项工作的第一个目的是基于负责 ATP 结合的氨基酸残基的鉴定，揭示蛋白质构象变化的机制。特别是，鉴定与 ATP γ 位磷酸盐相互作用的残基对于了解蛋白质的激活机制至关重要。第二个目的是确定蛋白质的结构域以调节其活性。为此，由于过度起始的发生，对带有野生型等位基因的细胞表现出致命性的 dnaA 突变体将被分离，并对突变蛋白进行生化分析。结果和讨论：使用 ATP 类似物进行亲和标记，其中包含在γ位上修饰磷酸盐并测定与该类似物共价结合的氨基酸残基表明Lys-415残基与ATP的γ磷酸盐相互作用。接下来，通过定点诱变将该残基替换为其他残基，并纯化这种突变蛋白。对这些的分析表明，Lys 残基对于 ATP 激活蛋白质是必需的（手稿正在准备中）。在第二个项目中，通过在 dnaA 顺反子中引入随机突变，我们获得了三个 dnaA 突变基因，这些基因对携带野生型等位基因的细胞显示出致命性。测序表明突变出现在 DnaA 蛋白的 ATP 结合域和 DNA 结合域之间的不同区域。此处分离的突变体 DnaA 的纯化和表征表明，该突变体蛋白缺乏 ATP 亲和力，但维持复制活性。这些结果表明这个独特区域的构象对于 DnaA 功能调节的重要性，是蛋白质结构功能关系研究的一个关键发现。