SYNTHESIS AND ASSEMBLY OF THE E COLI H+-ATPASE

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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.