SYNTHESIS AND ASSEMBLY OF THE E COLI H+-ATPASE

质子转运的ATPase在体内合成大部分的ATP 生物系统，具有非常相似的结构和功能 在细菌、植物和动物中。拟议的研究将 研究控制合成和组装的因素 大肠杆菌ATPase，以了解如何控制基因 表达影响组装和活性。装配和功能 还将研究由亚基组成的杂交ATPase 来自大肠杆菌和专性需氧细菌巨大芽孢杆菌。 大肠杆菌ATPase的亚基由下列基因编码 已经被证明是不同的UNC操纵子 从单个多顺反子mRNA体外翻译而来。框内 UNC基因与LacZ的融合将用于体内测量 染色体UNC基因的表达及检测 自体调控UNC基因表达的机制。 将开发一种系统来诱导UNC基因依赖的致死 质子渗透性，然后该系统将被用于研究 质子通道组装的生物化学。 大肠杆菌-巨型芽孢杆菌ATPase杂合复合体的形成 当E.coliUNC突变株与克隆株互补时 巨型芽孢杆菌ATPase DNA。为了进一步加深对 植物体内ATPase亚基的一级结构及其相互作用 复合体，剩下的巨大芽孢杆菌ATPase基因将是 克隆并测序。通过构建含有以下成分的质粒 巨型芽孢杆菌基因的各种组合和检测 每个质粒补充大肠杆菌突变体的能力， 外源亚基组装的物种特异性要求 将被确定为这些杂化络合物。另外，由于这些 ATPase可以将质子传导与ATP合成偶联，但不能 水解，同样的实验将确定亚基 参与了能量耦合。最后，杂交亚单位将是 构建用于确定单个ATPase的哪些部分 亚基，甚至哪些氨基酸残基参与其中 能量耦合和亚基-亚基相互作用 集合。

Proton-translocating ATPases synthesize most of the ATP in biological systems, and have a very similar structure and function in bacteria, plants, and animals. The proposed research will investigate the factors controlling the synthesis and assembly of the E.coli ATPase in order to understand how control over gene expression affects assembly and activity. Assembly and function will also be studied for a hybrid ATPase consisting of subunits from both E.coli and the obligate aerobe Bacillus megaterium. The subunits of the E.coli ATPase are coded for by the genes of the unc operon which have been shown to be differentially translated in vitro from a single polycistronic mRNA. In-frame fusions of unc genes to lacZ will be used to measure in vivo expression of chromosomal unc genes and to determine the mechanism of autogenous control over unc gene expression. A system will be developed to induce unc gene-dependent lethal proton permeability, and that system will then be used to study the biochemistry of assembly of the proton channel. Hybrid E.coli-B.megaterium ATPase complexes can be formed when E.coli unc mutants are complemented with cloned B.megaterium ATPase DNA. To further the understanding of the primary structures of ATPase subunits and their interactions in the complex, the remaining B.megaterium ATPase genes will be cloned and sequenced. By constructing plasmids containing various combinations of B.megaterium genes and testing the ability of each plasmid to complement E.coli mutants, the species-specific requirements for assembly of foreign subunits into these hybrid complexes will be determined. Also, since these ATPases can couple proton conduction to ATP synthesis but not hydrolysis, the same experiments will determine the subunits involved in energy coupling. Last, hybrid subunit will be constructed to determine which parts of individual ATPase subunits, and even which amino acid residues, are involved in energy coupling and in subunit-subunit interactions during assembly.