A series of interconnected studies in the model prokaryote Escherichia coli will address several incompletely understood facets of the trp and tyr regulatory systems. The focus of interest will be a group of specific interactions between macromolecules: protein-protein, protein- DNA, and protein-protein-DNA. The techniques to be employed will be those of molecular biology, microbial genetics, protein biochemistry and immunochemistry. The genes of the trp regulon are negatively controlled at the transcriptional level by the Trp repressor protein an alpha2 homodimer (MR per monomer, 12,000). The ability of this protein to associate with operator targets in duplex DNA requires a conformational change that is normally mediated by L-tryptophan. E. coli also produces a protein, designated TrbA, that enhances the rate of formation and/or the stability of complexes between the trp operator and the Trp holorepressor. The TrbA protein, an alpha2 homodimer (Mr per monomer, 22,000) is produced predominantly during the stationary phase of the growth cycle. Through a more detailed analysis of the molecular physiology of the Trp repressor-TrbA system, we expect to acquire a deeper understanding of how transcriptional control in the trp system is modulated throughout the growth cycle. We will test the proposition that the TrbA protein is important in communication gene regulatory information between actively dividing cells and cells in the stationary phase of growth. The mtr gene encodes a single-component membrane permease (414 amino acids) whose role is to mediate the concentrative uptake of L-tryptophan. The spatial disposition of membrane-spanning segments and the identity of residues in Mtr protein important for correct ligand transport will be addressed using mutational methods. The mtr promoter is strongly repressed by the Trp repressor and induced by the TryR protein. How the TyrR protein (an alpha2 homodimer having 513 amino acids per subunit) functions as a transcriptional enhancer of the mtr gene is of particular interest, given the fact that this protein mainly down-regulates gene expression by inhibiting transcription from several promoters (aroG, aroP, aroL, aroF, tyrP). in a tyrosine-dependent fashion. Although a central segment of the TyrR protein, at the amino acid level, bears strong homology to a series of transcriptional enhances specific for the sigma54 form of RNA polymerase, only promoters transcribed by the sigma70 form of RNA polymerase are controlled by this protein. The structural and mechanistic basis of transcriptional enhancement by the TryR protein will be investigated using biochemical methods as well as conventional and site-directed mutagenesis. The TyrR protein probably enhances transcription by making contact with some part of the RNA polymerase molecule. We will investigate the chemical nature of these contacts and attempt to discover how such protein-protein contracts help polymerase begin transcription. Because there is extensive homology among the largest protein subunits of eukaryotic and prokaryotic RNA polymerases, such questions are of broad and general interest.
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