Trp repressor protein of Escherichia coli is an alpha 2 homodimer (M/r per monomer, 12,339) that forms stable noncovalent complexes with a number of operator targets in double-stranded DNA, provided that certain conformational changes, mediated by L-tryptophan, have first taken place. Using the techniques of molecular genetics, protein chemistry, in vitro segment-directed mutagenesis and NMR specifrometry, we will seek to further refine our knowledge of which amino acids within Trp repressor are important for dimerization, ligand binding, and operator recognition. Particular attention will be focused on helix D, an eight amino acid stretch that comprises one part of a """"""""helix- turn-helix motif"""""""" common to all operator-binding proteins. The trpR gene is one member of a group of a least eight genes that bear significant structural homology to one another, as determine in Southern blotting experiments with total genomic DNA. The remaining members of this gene family will be cloned and structurally characterized. Several observations point to the existence of post- transcriptional and/or post-translational mechanisms for controlling the intracellular levels of functionally active Trp repressor. The details of these mechanisms will be examined using biochemical and genetic approaches, with a view to clarifying the broader aspects of how cells maintain regulatory specificity by controlling the levels of proteins like Trp repressor that have the capacity to intrude in non-physiological ways into heterologous systems. This is an important issue, because when strains are engineered so as to produced greatly elevated levels of Trp repressor, one observes reduced expression of the phe, leu, ilv and thr operons. This repression has been traced to the ability of Trp repressor to bind with low affinity to trpO-like sequences lying within the promoters for the above-named operons.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM022131-11A2
Application #
3270959
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-05-01
Project End
1992-05-31
Budget Start
1987-06-01
Budget End
1988-05-31
Support Year
11
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Purdue University
Department
Type
Earth Sciences/Resources
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
Smith, H Q; Somerville, R L (1997) The tpl promoter of Citrobacter freundii is activated by the TyrR protein. J Bacteriol 179:5914-21
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
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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