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.
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