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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM038912-05
Application #
3295697
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-07-01
Project End
1993-06-30
Budget Start
1992-07-01
Budget End
1993-06-30
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
Schools of Arts and Sciences
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Wu, W F; Urbanowski, M L; Stauffer, G V (1993) MetJ-mediated regulation of the Salmonella typhimurium metE and metR genes occurs through a common operator region. FEMS Microbiol Lett 108:145-50
Cowan, J M; Urbanowski, M L; Talmi, M et al. (1993) Regulation of the Salmonella typhimurium metF gene by the MetR protein. J Bacteriol 175:5862-6
Wu, W F; Urbanowski, M L; Stauffer, G V (1992) Role of the MetR regulatory system in vitamin B12-mediated repression of the Salmonella typhimurium metE gene. J Bacteriol 174:4833-7
Mares, R; Urbanowski, M L; Stauffer, G V (1992) Regulation of the Salmonella typhimurium metA gene by the metR protein and homocysteine. J Bacteriol 174:390-7
Byerly, K A; Urbanowski, M L; Stauffer, G V (1991) The metR binding site in the Salmonella typhimurium metH gene: DNA sequence constraints on activation. J Bacteriol 173:3547-53
Byerly, K A; Urbanowski, M L; Stauffer, G V (1990) Escherichia coli metR mutants that produce a MetR activator protein with an altered homocysteine response. J Bacteriol 172:2839-43
Urbanowski, M L; Stauffer, G V (1989) Role of homocysteine in metR-mediated activation of the metE and metH genes in Salmonella typhimurium and Escherichia coli. J Bacteriol 171:3277-81
Urbanowski, M L; Stauffer, G V (1989) Genetic and biochemical analysis of the MetR activator-binding site in the metE metR control region of Salmonella typhimurium. J Bacteriol 171:5620-9