Studies on the lep and rnc operons of Escherichia coli have shown that, in addition to encoding signal peptidase I and ribonuclease III respectively, they also both encode novel GTP-binding proteins. These findings are of general interest because GTP-binding proteins play a central role in regulation of cell growth in eukaryotes. Such an observation has not been made in prokaryotes. In this proposal a detailed genetic analysis of lepA and era (genes encoding E. coli GTP-binding proteins) will be combined with an in depth biochemical analysis of their gene products. Gene replacement, localized random mutagenesis, and site directed mutagenesis will be performed to obtain mutants in the region of interest. Interesting mutants will be analyzed phenotypically and by DNA sequence analysis. High level expression vectors will be employed to obtain pure protein (both mutant and wild type) for biochemical analysis. GTP-binding and GTPase activity will be assayed under a wide range of conditions. Due to the similarities between LepA and elongation factors parallel mutants will be constructed in the gene encoding elongation Factor G. These studies will lead to a better understanding of the role of GTP- binding proteins in all biological systems. Long range directions will include X-ray crystallographic analysis of these proteins, and investigations aimed at determining how wide spread they are in prokaryotic genera. In an effort to gain insight into the function of the era gene and the rnc operon, experiments aimed at characterizing the rnc promoter and mRNA are proposed. In addition, mutational studies similar to those proposed above will be employed to elucidate amino acid residues required by Ribonuclease III to recognize and cleave double stranded RNA.
