The objective of the proposed research is to understand in molecular terms the complex control circuits that regulate formation of the one-carbon units essential for cellular methylation reactions. In Escherichia coli and Salmonella typhimurium the serine-glycine and glycine cleavage pathways, which generate these one-carbon units, are under both positive and negative control. A combination of genetic and biochemical techniques will be used to study the fused to the lac operon. These fusions will be used to isolated both cis- and trans-acting mutations with altered regulation by both the positive- and negative- acting control elements. Oligonucleotide-directed site-specific mutagenesis will be used to further define cis-acting sites involved in gene expression. Several gene involved in these pathways have been cloned (glyA gene, gcv structural genes and gcv repressor gene), and a number of biochemical procedures will be used to analyze these genes at the molecular level (DNA sequencing, DNaseI protection assays, S1 nuclease mapping, gel mobility shift assays). In addition, a cell-free system will be used to examine regulation of transcription and translation in vitro using plasmid DNA carrying lac fusions as templates. This system will provide an assay for purified regulatory proteins and small effector molecules that are directly involved in the activation and repression mechanisms. The complexity of the serine- glycine and glycine cleavage pathways, where both positive- and negative- acting regulatory components must interact to coordinate gene expression, also make it a good model system in which it examine the more general phenomenon of protein-protein and DNA-protein interactions promoting gene expression.
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