MECHANISM OF TRP GENE REGULATION BY TRAP-RNA RECOGNITION

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Post-transcriptional regulatory mechanisms play a role in gene expression in probably all organisms. TRAP of Bacillus subtilis regulates expression of the trpEDCFBA operon by transcription attenuation and translational control mechanisms. When activated by tryptophan, TRAP binds to a 5' stem-loop (5'SL) and 11 (G/U)AG repeats in the nascent trp leader transcript, thereby promoting transcription termination before RNA polymerase can reach the trp structural genes (attenuation). TRAP binding also promotes formation of an RNA structure that inhibits trpE translation. These mechanisms will be further analyzed to gain a better understanding of B. subtilis trp operon regulation and the diversity of post-transcriptional regulatory mechanisms. Since several human disorders are caused by sequestration of triplet repeat RNA-binding proteins, combined with the findings that expression of HIV and several oncogenes is regulated by attenuation, results from these studies will indirectly contribute to improving human health. The role that the 5'SL plays in trp operon expression will be investigated. The amino acid residues of TRAP that interact with the 5'SL, as well as the 5'SL nucleotides that interact with TRAP will be determined by TRAP-5'SL RNA binding studies. The possibility that the 5'SL increases the rate of TRAP-trp leader RNA association will also be examined by performing binding assays. In addition, in vitro and in vivo experiments will be performed to determine if the 5'SL serves as an RNA polymerase pause signal and/or an mRNA instability determinant. The mechanism of translational control mediated by TRAP-dependent formation of the trpE Shine-Delgarno (SD) blocking hairpin will also be examined. In vivo expression studies will be carried out to determine if the 5'SL participates in this mechanism. In vivo expression, in vitro translation, and mRNA half-life experiments will also be performed to determine if formation of the trpE SD blocking hairpin regulates expression of the downstream genes via translational coupling, transcriptional polarity and/or decreasing mRNA stability. In addition, experiments will be carried out to determine if translational control requires a higher tryptophan concentration than is required for transcription attenuation. Finally, the possible role of an RNA pseudoknot in regulating translation and/or mRNA stability of the trp operon will be determined by several genetic and biochemical approaches.