The goal of this proposal is to understand at the molecular level, the physiology of coordinate gene regulation, a process that occurs in essentially all organisms. Using the trp regulon of the bacterium. Escherichia coli as a model system, we will correlate biochemical and genetic data to the physiological events occurring within the cell. Because of the recent advances in molecular and genetic methods, we are approaching the stage that a quantitative understanding of how the cell coordinately regulate sets of operons via common regulatory signals becomes experimentally possible. We wish to understand how a cell designs the transcription and translation control element of each operon in the regulon to insure that a desire level of gene expression occurs in response to the common intracellular signal. By knowing the essential parameters, we should be able to design regulatory elements that respond in a predictable fashion to the sensitivity and magnitude of repression desired. Comparative in vivo and in vitro analysis of the trp, trpR and aroH regulatory elements will be done. Mutations in each of the promoters and operators will be generated and analyzed to determine how repression is affected in response to control by trp repressor and corepressor. Contribution of the trpR and aroH leader regions on the expression of the respective operons will be examined. Evaluation of a multiple trp repressor binding site model for the trp and aroH operators will be examined to establish their role in in vivo operon expression. Coordinate gene regulation processes occur in virtually all organisms throughout nature whether it be for the control of intermediary or catabolic functions involved in bacterial metabolism, or in analogous processes in eucaryotic organisms. This system is also a simple model for understanding more complex ligand regulated gene systems in higher organisms including steroid hormonal control as well as developmentally controlled processes. A thorough quantitative analysis of the variables affecting transcription of the three operons of the trp region should make it possible to understand how a single repressor species can effect different levels of regulation in response to a single intracellular signal. Whereas understanding the individual components of these regulatory processes is required, it may not be sufficient for understanding how each contributes to the integrated physiology within the normal context of the living cell. The trp regulon offers an exceptional model for extending the study of gene regulation to this next level, and it is hoped that emergent properties will result.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029456-09
Application #
3277042
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1981-09-29
Project End
1993-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
9
Fiscal Year
1990
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
Evans, J N; Arvidson, D N; Gunsalus, R P et al. (1992) Multinuclear NMR studies of the trp-repressor. Biochim Biophys Acta 1160:156-62
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