SYNTHESIS, ASSEMBLY & FUNCTION OF BACTERIAL H--ATPASES

质子转运ATP酶合成大部分ATP， 生物系统，并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ATP酶，以了解如何控制基因 表达影响组装和活性。 组装和功能 还将研究由亚基组成的杂合ATP酶 来自大肠杆菌和专性需氧菌巨大芽孢杆菌。 大肠杆菌ATP酶的亚基由以下基因编码： 已经显示UNC操纵子 从单个多顺反子mRNA体外翻译。 框内 将使用unc基因与lacZ的融合物来体内测量 染色体unc基因的表达，并确定 自身调控unc基因表达的机制。 将开发一种系统来诱导unc基因依赖性致死 质子渗透性，然后该系统将用于研究 质子通道组装的生物化学。 可以形成杂合大肠杆菌-巨大芽孢杆菌ATP酶复合物 当大肠杆菌unc突变体与克隆的 B.megaterium ATPase DNA。 为了进一步了解 ATP酶亚基的一级结构及其相互作用 在复合物中，剩余的B.megaterium ATP酶基因将被 克隆和序列测定 通过构建含有 B.megaterium基因的各种组合，并测试 每个质粒互补大肠杆菌突变体的能力， 外来亚基组装的物种特异性要求 将确定这些混合复合物。 此外，由于这些 ATP酶可以将质子传导与ATP合成偶联， 水解，同样的实验将确定亚基 参与能量耦合。 最后，混合亚基将被 构建来确定单个ATP酶的哪些部分 亚基，甚至是哪些氨基酸残基，参与了 能量耦合和亚基-亚基相互作用 组装件.

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.