SYNTHESIS AND ASSEMBLY OF THE E COLI H+-ATPASE

质子转移三磷酸腺苷酶催化脑内氧化磷酸化 线粒体、叶绿体和细菌。这项工作将考察 参与生物发生(合成和组装)的因素 大肠杆菌的质子转运ATPase。尽管8号病毒的基因 该ATPase的亚基在操纵子(UNC)中以相等的数量排列 操纵子)，这些基因的表达反映了这样一个事实 在ATPase复合体中以不同的数量存在。建议数 研究将调查其中几个基因是如何表达的 以及该对照的变化如何影响ATPase和E.Coli. 携带着这些突变。 ATPase基因的表达将从两个方面进行研究。蛋白质产品 ATPase基因控制的体外转录-翻译系统的构建 克隆到不同的质粒上将被定量，β-半乳糖苷酶 活性将通过ATPase基因和lac Z之间的基因融合来衡量。 控制UNC基因表达的区域突变将是 使用重组DNA技术在体外构建或将被分离 通过筛选β-半乳糖苷酶在体内的高表达 UNC-lac基因融合。携带这种突变的细胞的表型将 旨在将亚基表达的解除调控与 ATPase的功能结构。信使核糖核酸二级结构的作用 在控制不同基因表达方面的体外研究将使用 人工合成的寡核苷酸来破坏这种结构。 此外，F1 ATPase组装过程中的初始事件将是 用抗人免疫球蛋白抗体在体外系统中进行研究 单个亚基以沉淀形成的复合体 抗原亚基和其他F1亚基。在这些研究中，体外试验 转录-翻译系统可以在三个层面上进行操作：1) 基因将被表达，2)哪个亚基将被沉淀，以及3) 哪些纯化的亚基可以添加到反应中以影响 形成复合体。

Proton-translocating ATPases catalyze oxidative phosphorylation in mitochondria, chloroplasts and bacteria. This work will examine the factors involved in the biogenesis (synthesis and assembly) of the proton-translocating ATPase of E. coli. Although the genes for the 8 subunits of this ATPase are arranged in equal numbers in an operon (the unc operon), the expression of those genes reflects the fact that the subunits are present in different numbers in the ATPase complex. The proposed research will investigate how the expression of several of these genes is controlled and how changes in that control affect the ATPase and E. coli carrying those mutations. Expression of ATPase genes will be studied two ways. The protein products of an in vitro transcription-translation system directed by ATPase genes cloned onto different plasmids will be quantitated, and Beta-galactosidase activity will be measured from gene fusions between ATPase genes and lac Z. Mutations in regions controlling expression of unc genes will be constructed in vitro using recombinant DNA techniques or will be isolated in vivo by screening for increased expression of Beta-galactosidase from unc-lac gene fusions. The phenotypes of cells carrying such mutations will be studied in an effort to relate deregulation of subunit expression to the structure of function of the ATPase. The role of mRNA secondary structure in controlling expression of different genes will be studied in vitro using synthetic oligonucleotides to disrupt such structures. Also, the initial events in the assembly of the F1 ATPase will be investigated in the in vitro system by using antibodies raised against individual subunits to precipitate complexes formed between the antigen-subunit and other F1 subunits. For these studies, the in vitro transcription-translation system can be manipulated at 3 levels: 1) which genes are to be expressd, 2) which subunit will be precipitated, and 3) which purified subunits can be added to the reactions to affect the formation of complexes.