In the fluorescent pseudomonads an independent 3-gene cluster is responsible for producing tryptophan synthase. In addition to the 2-gene operon encoding the enzyme's alpha and beta polypeptides, a divergently transcribed activator gene (trpI) belonging to a new family of bacterial regulators mediates induction of the enzyme by its substrate, indoleglycerol phosphate. We will examine the mechanism of this activation through: purification of the trpI gene product to homogeneity; identification of its DNA-binding domain by evolutionary comparison, directed mutagenesis and partial proteolysis; identification of its chromosomal recognition site by evolutionary comparison, deletion analysis and both spontaneous and directed mutagenesis; development of a filter-binding assay to complement the available footprinting and gel retardation assays; making fusions to place other genes (trpEGDC and xylE) under trpI control. In addition we will investigate the generality of the recently described """"""""tunnel"""""""" connecting the two active sites in the three-dimensional structure of Salmonella typhimurium tryptophan synthase by examining its relationship to an unusual class of """"""""repairable"""""""" trpB mutants studied earlier. We will: clone and sequence most of the existing mutants (using the polymerase chain reaction) to establish the nature and location of the amino acid substitution; overproduce selected mutant proteins by use of a high-expression vector; characterize the extent of """"""""repairability"""""""" by the trpA gene product and ammonium ions; correlate the enzymatic defect with the ability of normal and borohydride-reduced beta 2 subunits to bind indole.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI020279-09
Application #
3129850
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1983-07-01
Project End
1994-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
9
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
Subbramanian, Ramu A; Moriya, Chikaya; Martin, Kristi L et al. (2004) Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity. Nat Biotechnol 22:1429-34
Pineiro, S; Olekhnovich, I; Gussin, G N (1997) DNA bending by the TrpI protein of Pseudomonas aeruginosa. J Bacteriol 179:5407-13
Chang, M; Crawford, I P (1991) In vitro determination of the effect of indoleglycerol phosphate on the interaction of purified TrpI protein with its DNA-binding sites. J Bacteriol 173:1590-7
Crawford, I P; Han, C Y; Silverman, M (1991) Sequence and features of the tryptophan operon of Vibrio parahemolyticus. DNA Seq 1:189-96
Bae, Y M; Stauffer, G V (1991) Mutations that affect activity of the Rhizobium meliloti trpE(G) promoter in Rhizobium meliloti and Escherichia coli. J Bacteriol 173:5831-6
Bae, Y M; Stauffer, G V (1991) Genetic analysis of the attenuator of the Rhizobium meliloti trpE(G) gene. J Bacteriol 173:3382-8
Bae, Y M; Crawford, I P (1990) The Rhizobium meliloti trpE(G) gene is regulated by attenuation, and its product, anthranilate synthase, is regulated by feedback inhibition. J Bacteriol 172:3318-27
Chang, M; Crawford, I P (1990) The roles of indoleglycerol phosphate and the TrpI protein in the expression of trpBA from Pseudomonas aeruginosa. Nucleic Acids Res 18:979-88
Crawford, I P (1989) Evolution of a biosynthetic pathway: the tryptophan paradigm. Annu Rev Microbiol 43:567-600
Chang, M; Hadero, A; Crawford, I P (1989) Sequence of the Pseudomonas aeruginosa trpI activator gene and relatedness of trpI to other procaryotic regulatory genes. J Bacteriol 171:172-83

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