RECA PROTEIN STRUCTURE, FUNCTION, AND CONTROL

我们的目标是阐明结构的分子特征， E.大肠杆菌recA蛋白，并将这些结构细节与酶促 蛋白质的特性。 将采取的办法包括 结合生物化学修饰研究、重组DNA技术 和“经典”微生物遗传学。 recA蛋白的生物化学研究包括直接光链接 将DNA连接到recA蛋白的多核苷酸结合位点。 识别 与DNA共价交联的recA蛋白的区域将是 使用胰蛋白酶消化、HPLC和氨基酸分析完成。 使用 一种类似的方法，recA蛋白的ATP结合位点已经被鉴定， 我们现在希望鉴定ATP与之结合的氨基酸残基， 共价连接。 这将通过进一步的蛋白水解来实现。 修饰肽的消化和氨基酸序列分析。 使用 光亲和标记和胰蛋白酶图谱技术，我们将研究 该ATP结合结构域在五种异源recA中的保守性 来自P. vulgaris、E. carotovora，S.鼠伤寒沙门氏菌（S. flexneri和E.大肠杆菌B/r. 这些异源蛋白的基因具有 克隆到E. coli K12中表达，并在 合适的遗传背景。 这一比较分析将提供 关于recA蛋白ATP相互作用的性质的有价值的信息。 通过生物化学修饰研究获得的信息， recA蛋白将允许更详细的功能分析， 应用定点诱变技术， recA结构基因。 Ba 131缺失和 将采用阿糖胞苷定向诱变来修饰那些 recA蛋白质的区域对于底物结合是重要的。 我们将 然后分析这些修饰的效果或recA的功能， 蛋白质在体外和体内。 关于recA结构域的其他信息将通过DNA测序获得。 序列分析表明，所克隆的recA基因分别来自普通对虾、E.胡萝卜软腐菌， S.鼠伤寒沙门氏菌（S. flexneri和E.大肠杆菌B/r. 克隆人的能力 基因来弥补E. coliK 12 recA突变表明 在这些异源中保护重要的功能结构域 proteins.

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.