Despite the importance of one-carbon units in cell physiology, our understanding of the regulation of their biosynthesis and utilization is limited. The long term objective of this research project is to define the molecular mechanisms for regulation of the genes involved in their production and utilization in both E. coli and S. typhimurium. We have taken a genetic and a biochemical approach to study one-carbon metabolism. New selection procedures have been developed to facilitate the isolation and characterization of regulatory mutations. In addition, the lac operon of E. coli has been fused to the glyA and serB promoters of E. coli and the glyA, metE, metB and metJ promoters of S. typhimurium. Fusions of the S. typhimurium metH and metF promoters and the E. coli gcv promoter(s) to the lac operon will be constructed. Mutations that alter expression of the lac genes will be isolated and characterized in an effort to identify gene products (e.g., activator or repressor proteins) and DNA sequence elements (e.g., promoters, operators or activator binding sites) that regulate expression of the genes involved in one-carbon metabolism. Genetic techniques (transduction, partial diploid analysis, etc.) will be used to locate the sites of the mutations on the E. coli and S. typhimurium linkage maps and to determine the effects of the mutations in trans. Several genes involved in one-carbon metabolism have been cloned (glyA, gcv, serB, metJ, metB, metE, metH and metF), and a number of biochemical procedures have been utilized to analyze these genes at the molecular level (DNA sequencing, DNAase I footprinting, S1 nuclease mapping, etc.). In addition, a cell-free transcription-translation system will be used to examine regulation in vitro, using plasmid DNA carrying the lac fusions as templates. Ultimately, this system will provide an assay for cellular components directly involved in the regulation of these genes. Together, the genetic and biochemical analyses will allow us to determine the nature of the controls acting on genes directly involved in one-carbon production and utilization, and the importance of these pathways in cell physiology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026878-08
Application #
3274328
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1979-07-01
Project End
1990-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
8
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Urbanowski, M L; Stauffer, L T; Stauffer, G V (2000) The gcvB gene encodes a small untranslated RNA involved in expression of the dipeptide and oligopeptide transport systems in Escherichia coli. Mol Microbiol 37:856-68
Wonderling, L D; Urbanowski, M L; Stauffer, G V (2000) GcvA binding site 1 in the gcvTHP promoter of Escherichia coli is required for GcvA-mediated repression but not for GcvA-mediated activation. Microbiology 146 ( Pt 11):2909-18
Jourdan, A D; Stauffer, G V (1999) Genetic analysis of the GcvA binding site in the gcvA control region. Microbiology 145 ( Pt 8):2153-62
Wonderling, L D; Stauffer, G V (1999) The cyclic AMP receptor protein is dependent on GcvA for regulation of the gcv operon. J Bacteriol 181:1912-9
Jourdan, A D; Stauffer, G V (1999) GcvA-mediated activation of gcvT-lacZ expression involves the carboxy-terminal domain of the alpha subunit of RNA polymerase. FEMS Microbiol Lett 181:307-12
Stauffer, L T; Stauffer, G V (1999) Role for the leucine-responsive regulatory protein (Lrp) as a structural protein in regulating the Escherichia coli gcvTHP operon. Microbiology 145 ( Pt 3):569-76
Stauffer, L T; Stauffer, G V (1998) Roles for GcvA-binding sites 3 and 2 and the Lrp-binding region in gcvT::lacZ expression in Escherichia coli. Microbiology 144 ( Pt 10):2865-72
Stauffer, L T; Stauffer, G V (1998) Spacing and orientation requirements of GcvA-binding sites 3 and 2 and the Lrp-binding region for gcvT::lacZ expression in Escherichia coli. Microbiology 144 ( Pt 5):1417-22
Jourdan, A D; Stauffer, G V (1998) Mutational analysis of the transcriptional regulator GcvA: amino acids important for activation, repression, and DNA binding. J Bacteriol 180:4865-71
Ghrist, A C; Stauffer, G V (1998) Promoter characterization and constitutive expression of the Escherichia coli gcvR gene. J Bacteriol 180:1803-7

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