We hope to gain in a greater understanding of trp operon regulation at the repressor-operator level. Our immediate goals are to first expand and improve upon the present isolation procedure for obtaining pure trp aporepressor. This will be acomplished by constructing new plasmid bearing strains which produce elevated amounts of aporepressor. The purification procedure will then be enlarged in capacity to yield suitable amounts of repressor for the following proposed studies. We will further characterize the trp aporepressor protein in detail to determine its native size, isoelectric point, the UV-visible spectra and spectrofluorometric properties. Interaction of the trp repressor with the three operators it binds (trp, trpR, aroH) will be examined by the examined by the well established procedures of DNase """"""""foot-printing"""""""" and DMS methylation of the operator DNA fragments in the presence and absence of bound repressor. Using trpR containing DNA fragments in vitro mutagenesis will be carried out to generate regulatory and structurely altered repressor genes. These studies will be designed to obtain altered operators that may result in operon constitutivity in addition to repressor proteins altered for corepressor ligand and operator recognition. The availability of pure aporepressor should permit a detailed study of repressor-operator interaction. Using the existing in vitro transcription repressible assay it should be possible to measure dissociation constants for the trpR and aroH operators in addition to the binding by altered repressors. Traditional nitrocellulose filter binding assays experiments for trp repressor binding studies, previously unsuccessful will be reexamined now that milligram amounts of pure repressor are available. By extending studies on the trp repressor-operator we hope to gain a more detailed understanding of gene control.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029456-05
Application #
3277039
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-09-29
Project End
1988-06-30
Budget Start
1986-07-01
Budget End
1987-06-30
Support Year
5
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Cotter, P A; Gunsalus, R P (1992) Contribution of the fnr and arcA gene products in coordinate regulation of cytochrome o and d oxidase (cyoABCDE and cydAB) genes in Escherichia coli. FEMS Microbiol Lett 70:31-6
Melville, S B; Gunsalus, R P (1990) Mutations in fnr that alter anaerobic regulation of electron transport-associated genes in Escherichia coli. J Biol Chem 265:18733-6
Cotter, P A; Chepuri, V; Gennis, R B et al. (1990) Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product. J Bacteriol 172:6333-8
Kumamoto, A A; Miller, W G; Gunsalus, R P (1987) Escherichia coli tryptophan repressor binds multiple sites within the aroH and trp operators. Genes Dev 1:556-64
Grove, C L; Gunsalus, R P (1987) Regulation of the aroH operon of Escherichia coli by the tryptophan repressor. J Bacteriol 169:2158-64
Bass, S; Sugiono, P; Arvidson, D N et al. (1987) DNA specificity determinants of Escherichia coli tryptophan repressor binding. Genes Dev 1:565-72
Arvidson, D N; Bruce, C; Gunsalus, R P (1986) Interaction of the Escherichia coli trp aporepressor with its ligand, L-tryptophan. J Biol Chem 261:238-43
Gunsalus, R P; Miguel, A G; Gunsalus, G L (1986) Intracellular Trp repressor levels in Escherichia coli. J Bacteriol 167:272-8