Analysis of Regulatory Mechanism Controlling Tryptophan Metabolism in Bacteria

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0093023 Charles YanofskyThis research will focus on three principle objectives. The first objective is to characterize the YczA regulatory protein of Bacillus subtilis and determine how it inactivates the TRAP regulatory protein. The TRAP protein regulates transcription of six of the tryptophan biosynthetic genes, it regulates the seventh trp gene translationally. The YczA protein is produced by the yczA-ycbK operon, an operon of previously unknown function. This operon is regulated by transcription attenuation; it is transcriptionally activated in response to the accumulation of uncharged tRNATrp. The second objective is to continue studies on the detailed features of the mechanism of transcription attenuation used by E. coli and other bacterial species to regulate expression of the degradative tryptophanase(tna) operon. Tryptophan addition to E. coli cultures induces expression of the tna operon. Induction leads to inhibition of cleavage of a leader peptidyl-tRNA and this inhibition stalls the translating ribosome. The stalled ribosome prevents Rho mediated transcription termination in the leader region of the tna operon. The PI's laboratory has successfully reproduced the regulatory features for this operon in vitro and now plan to examine in detail exactly how peptidyl-tRNA cleavage is blocked. The third objective is to use DNA microarray analyses with the entire genome of Bacillus subtilis to determine which genes are selectively transcriptionally activated and transcriptionally inhibited in response to changes in tryptophan metabolism, as the PI recently did with the genome of E. coli. These findings should allow the comparison of expression of the genes of tryptophan metabolism in two microorganisms that are preferred subjects for experimental analysis.