In S. typhimurium cysteine biosynthesis is controlled by a combination of feedback inhibition of serine transacetylase and a system of positive gene regulation in which the cysteine precursor O-acetylserine is an internal inducer and the cysB protein effects expression of cys genes. Wild type and mutant forms of cysB protein that cause altered expression of the cysteine region will be purified and their primary structure deduced from DNA sequencing of their genes. Interactions of these proteins will be characterized in a cell-free transcription system using cloned promoter regions from several wild type and mutant cys genes. These studies will provide insight into the molecular mechanism of positive gene control. In studies on mammalian sulfur metabolism, human lymphocyte S-adenosylmethionine synthetase will be characterized both kinetically and structurally. A complete kinetic solution for this enzyme will then be used to predict factors that may be involved in its in vivo regulation, and cultured cell systems will be employed to test such hypotheses. Intracellular levels of S-adenosylmethionine and metabolites thought to regulate the synthesis of this methylation-donor will be measured together with rates of S-adenosylmethionine utilization. Control mechanisms will be evaluated in cells perturbed by various nutritional and chemical means. In vivo alterations in the protein subunit structure of S-adenosylmethionine synthetase will also be evaluated for regulatory significance.
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