Genetic and Biochemical Studies of the Tryptophan Operon

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Studies will continue on the regulatory-metabolic network that modulates uptake, synthesis and utilization of tryptophan in Escherichia coli. The effects of nutritional shifts will be examined in wild type, and in mutants lacking specific regulatory functions. Global effects of the TyrR protein will be explored. in vitro band shift analyses will be performed with RNA polymerase, trp superrepressor, and various target operators, to determine the basis of the differential in vivo regulation of transcription initiation in the four operons controlled by the trp repressor of E. coli. in vivo experiments will be performed to determine the effects of replacing the normal repressor by a slowly dissociating superrepressor. Second and third site revertants will be isolated in tryptophan synthetase a mutants altered in catalytic residues; residue changes should reveal important features of the active site of this protein. The basis of the defects in Crawford's trpB repairable mutants will be examined. The amino and carboxy terminal segments of the tryptophan synthetase chains of E. coli will be systematically altered in an effort to explain the order of gene fusion that occured during evolution of the fused tryptophan synthetase structural gene of fungi. %%% Much of what is known about metabolism of the amino acid tryptophan in bacteria is due to the work of this laboratory. Although many details of the complex network of regulation controlling tryptophan synthesis and utlization can now be explained, the problem still offers many avenues of study that will provide information concerning a wide range of fundamental biochemical and genetic processes.