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.
