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.
