RECA PROTEIN STRUCTURE, FUNCTION, AND CONTROL

我们的目标是阐明结构的分子特征 大肠杆菌recA蛋白并将这些结构细节与酶促反应联系起来 蛋白质的性质。 应采取的方法包括 生化修饰研究、重组DNA技术的结合 和“经典”微生物遗传学。 reCA 蛋白的生化研究包括直接光连接 DNA 与recA 蛋白的多核苷酸结合位点的结合。 鉴定 与DNA共价交联的recA蛋白区域的数量将是 使用胰蛋白酶消化、HPLC 和氨基酸分析完成。 使用 类似的方法已经确定了recA蛋白的ATP结合位点 现在我们希望确定 ATP 所在的氨基酸残基 共价连接。 这将通过进一步的蛋白水解来完成 修饰肽的消化和氨基酸序列分析。 使用 我们将研究光亲和标记和胰酶图谱技术 五个异源recA中该ATP结合域的保守性 源自 P. vulgaris、E. carotovora、S.typhimurium、S. 福氏菌和大肠杆菌 B/r。 这些异源蛋白质的基因 被克隆到大肠杆菌 K12 中并在 适当的遗传背景。 这种比较分析将提供 关于recA 蛋白ATP 相互作用性质的有价值的信息。 通过领域的生化修饰研究获得的信息 recA 蛋白将允许通过以下方式进行更详细的功能分析 定点诱变技术的应用 位于recA结构基因的区域。 Ba131 缺失和 寡核苷酸定向诱变将被用来修饰那些 对于底物结合很重要的recA 蛋白区域。 我们会 然后分析这些修改的效果或者recA的功能 体外和体内的蛋白质。 关于recA结构域的附加信息将通过DNA获得 对来自 P. vulgaris、E. carotovora 的克隆 recA 基因进行序列分析， 鼠伤寒沙门氏菌、福氏沙门氏菌和大肠杆菌 B/r。 克隆人的能力 补充大肠杆菌 K12 reA 突变缺陷的基因表明 这些异源之间重要功能域的保守 蛋白质。

Our goals are to elucidate the molecular features of the structure, of the E. coli recA protein and relate these structural details to the enzymatic properties of the protein. The approaches to be taken include a combination of biochemical modification studies, recombinant DNA techniques and 'classical' microbial genetics. Biochemical investigations of recA protein include the direct photolinking of DNA to the polynucleotide binding site of recA protein. Identification of the regions of recA protein covalently crosslinked to DNA will be accomplished using tryptic digestion, HPLC and amino acid analyses. Using a similar approach the ATP binding site of recA protein has been identified and we now wish to identify the amino acid residue(s) to which ATP is covalently linked. This will be accomplished by further proteolytic digestion of the modified peptide and amino acid sequence analysis. Using photoaffinity labeling and tryptic mapping techniques we will investigate the conservation of this ATP binding domain among five heterologous recA proteins derived from P. vulgaris, E. carotovora, S. typhimurium, S. flexneri and E. coli B/r. The genes for these heterologous proteins have been cloned into E. coli K12 and are expressed at high levels in appropriate genetic backgrounds. This comparative analysis will provide valuable information on the nature of recA protein ATP interaction. The information gained by biochemical modification studies of domains of recA protein will permit a more detailed functional analysis through the application of site-directed mutagenesis techniques to the appropriate region of the recA structural gene. Both Ba131 deletion and oligonucleotide-directed mutagenesis will be employed to modify those regions of recA protein that are important for substrate binding. We will then analyze the effects of these modifications or the functions of recA protein both in vitro and in vivo. Additional information on the domains of recA will be obtained by DNA sequence analysis of the cloned recA genes from P. vulgaris, E. carotovora, S. typhimurium, S. flexneri and E. coli B/r. The ability of the cloned genes to complement defects of an E. coli K12 recA- mutation indicates conservation of important functional domains among these heterologous proteins.