GENETIC ANALYSIS OF THE SECRETORY APPARATUS OF E COLI

E.大肠杆菌，它阻止合成 已分离出的蛋白质。 在真核生物中， 信号序列突变可以识别分泌的一种成分， 装置;这种认识导致逮捕的翻译 输出蛋白质。 合成直到被捕获的翻译 复合物与内质网（ER）膜接触。 的 e.大肠杆菌分泌突变体secC，分离为假回复突变体， 另一个分泌突变体secA。 在23摄氏度下，secC突变体阻止 运输蛋白质的合成。 在这个突变体中， 可以通过突变输出蛋白质的信号序列来逆转输出蛋白质的信号序列。 这种蛋白质。 这项建议有几个目的。 首先，输出蛋白质的突变， 特异性逆转secC中蛋白质的合成阻断 突变体，将被分离和测序。 这项工作应该定义 参与其合成的输出蛋白质的结构域。 第二、 secC突变体假回复突变体的分离应鉴定基因 其蛋白质产物与secC基因产物相互作用。 三是 肩关节secC基因敲除突变体分离及表型分析 最终解开secC蛋白的作用。 这个基因敲除突变体 将通过使用Tn5诱变来构建。 的进一步表征 也将进行secC突变体。 抗SecA抗体 蛋白质将用于确定SecC和SecA蛋白质是否 互动. 免疫沉淀和温度变化实验，在 存在阻止蛋白质合成起始的药物， 应该确定合成中的阻断是否是位点特异性的。 效果 也将评估secC突变对SecC蛋白的影响。 蛋白质分泌是一个重要的细胞过程。 的研究 E.大肠杆菌可能会加强我们对此的理解 在所有生物体中。

Mutants in the secretory apparatus of E. coli, which block the synthesis of exported proteins, have been isolated. In eukaryotes, it has been shown that signal sequence mutations can recognize a component of the secretory apparatus; this recognition results in an arrest in the translation of exported proteins. Synthesis is not resumed until the arrested translation complex comes in contact with the endoplasmic reticulum (ER) membrane. The E. coli secretory mutant, secC, was isolated as a pseudorevertant of another secretion mutant, secA. At 23 degrees C, the secC mutant blocks the synthesis of transported proteins. In this mutant, the synthesis block of an exported protein can be reversed by mutating the signal sequence of that protein. This proposal has several aims. First, mutations in an exported protein, which specifically reverse the synthesis block of that protein in the secC mutant, will be isolated and sequenced. This work should define the domains of an exported protein that are involved in its synthesis. Second, the isolation of pseudorevertants of the secC mutant should identify genes whose protein products interact with the secC gene product. Third, the isolation and phenotypic analysis of a knock-out mutant in secC should ultimately unravel the role of the secC protein. This knock-out mutant will be constructed by using Tn5 mutagenesis. Further characterization of the secC mutant will also be done. Antibody directed against the SecA protein will be used to determine whether the SecC and SecA proteins interact. Immunoprecitation and temperature shift experiments, in the presence of a drug that prevents the initiation of protein synthesis, should determine if the block in synthesis is site-specific. The effect that the secC mutation has on the SecC proteins will also be evaluated. Protein secretion is an essential cellular process. Studies on the mechanism of secretion in E. coli may strengthen our understanding of this process in all organisms.