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 蛋白质的性质。将采取的方法包括 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键连接 DNA结合到recA蛋白的多核苷酸结合部位。鉴定 在recA蛋白与DNA共价交联的区域中 通过胰酶消化、高效液相色谱和氨基酸分析完成。vbl.使用 一种类似的方法已经确定了recA蛋白的ATP结合部位 现在我们要确定三磷酸腺苷的氨基酸残基(S) 共价连接的。这将通过进一步的蛋白质分解来实现。 对修饰后的多肽进行酶切和氨基酸序列分析。vbl.使用 我们将研究光亲和标记和胰酶图谱技术 该ATP结合域在五个异源RecA中的保守性 来源于普通拟青霉、胡萝卜拟青霉、鼠伤寒沙门氏菌、沙门氏菌的蛋白质。 Flexneri和E.coliB/r。这些异源蛋白的基因有 已克隆到E.ColiK12中，并在 合适的遗传背景。这一比较分析将提供 关于recA蛋白ATP相互作用性质的有价值的信息。 通过对结构域的生化修饰研究获得的信息 RecA蛋白将允许通过 定点诱变技术在家蚕中的应用 RecA结构基因的区域。Ba131缺失和 将使用寡核苷酸定向突变来修改这些 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RecA的功能 体外和体内的蛋白质。 有关recA结构域的更多信息将通过DNA获得 克隆的普通拟青霉、胡萝卜拟青霉recA基因序列分析 鼠伤寒沙门氏菌、福氏沙门氏菌和E.ColiB/r。 补充大肠杆菌K12 recA突变缺陷的基因表明 这些异源基因之间重要功能结构域的保守性 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.