The objective of the proposed research is to understand in molecular terms the mechanism(s) involved in activation of gene expression using the methionine pathway as a model system. In Escherichia coli and Salmonella typhimurium, the metE and metH genes are positively controlled by the metR gene product. In addition, the metE gene is negatively controlled by two independent repressors, the metJ and metH gene products. A combination of genetic and biochemical techniques will be used to study regulation of these genes. The metE, metH and metR control regions have been fused to the lac operon. The metE-lac and metH-lac fusions will be used to isolate mutants with altered regulation by the positive control system. The metR-lac fusion will be used to study regulation of the metR gene itself. Oligonucleotide site directed mutagenesis will be used to alter sites presumed to be involved in gene expression. In addition, a cell free system will be developed to examine regulation in vitro using plasmid DNA carrying the lac fusions as templates. This system will provide an assay for cellular components directly invovled in the activation mechanism. Becuase of the complexity of the methionine control system, where both positive and negative acting regulatory components must interact to coordinate gene expression, it is an excellent model system in which to examine the details of DNA-protein and protein-protein interactions promoting gene expression.
