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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM022131-17
Application #
2173873
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-05-01
Project End
1997-06-30
Budget Start
1994-07-01
Budget End
1995-06-30
Support Year
17
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Purdue University
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
072051394
City
West Lafayette
State
IN
Country
United States
Zip Code
47907
Wang, Y; Zhao, S; Somerville, R L et al. (2001) Solution structure of the DNA-binding domain of the TyrR protein of Haemophilus influenzae. Protein Sci 10:592-8
Kristl, S; Zhao, S; Knappe, B et al. (2000) The influence of ATP on the binding of aromatic amino acids to the ligand response domain of the tyrosine repressor of Haemophilus influenzae. FEBS Lett 467:87-90
Zhao, S; Zhu, Q; Somerville, R L (2000) The sigma(70) transcription factor TyrR has zinc-stimulated phosphatase activity that is inhibited by ATP and tyrosine. J Bacteriol 182:1053-61
Zhao, S; Somerville, R L (1999) Isolated operator binding and ligand response domains of the TyrR protein of Haemophilus influenzae associate to reconstitute functional repressor. J Biol Chem 274:1842-7
Zhu, Q; Zhao, S; Somerville, R L (1997) Expression, purification, and functional analysis of the TyrR protein of Haemophilus influenzae. Protein Expr Purif 10:237-46
Smith, H Q; Somerville, R L (1997) The tpl promoter of Citrobacter freundii is activated by the TyrR protein. J Bacteriol 179:5914-21
Chen, Y W; Dekker, E E; Somerville, R L (1995) Functional analysis of E. coli threonine dehydrogenase by means of mutant isolation and characterization. Biochim Biophys Acta 1253:208-14
Zhao, G P; Somerville, R L; Chitnis, P R (1994) Synechocystis PCC 6803 contains a single gene for the beta subunit of tryptophan synthase with strong homology to the trpB genes of Arabidopsis and maize (Zea mays L.). Plant Physiol 104:461-6
Cui, J; Somerville, R L (1993) A mutational analysis of the structural basis for transcriptional activation and monomer-monomer interaction in the TyrR system of Escherichia coli K-12. J Bacteriol 175:1777-84
Cui, J; Ni, L; Somerville, R L (1993) ATPase activity of TyrR, a transcriptional regulatory protein for sigma 70 RNA polymerase. J Biol Chem 268:13023-5

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