TRP RNA-BINDING ATTENUATION PROTEIN (TRAP)-TRP LEADER RNA INTERACTIONS MEDIATE TRANSLATIONAL AS WELL AS TRANSCRIPTIONAL REGULATION OF THE BACILLUS-SUBTILIS TRP OPERON

Expression of the Bacillus subtilis trpEDCFBA operon has been shown to be regulated by transcription attenuation in response to the availabi lity of L-tryptophan, Regulation is mediated by the tryptophan-activat ed frp RNA-binding attenuation protein, TRAP, the product of mtrB. For mation of mutually exclusive RNA antiterminator and terminator structu res within trp leader RNA determines whether transcription will termin ate in the leader region of the operon, Previous studies suggested tha t transcripts that escape termination are subject to translational reg ulation via the formation of a secondary structure that blocks ribosom e access to the trpE ribosome binding site. To assess the relative imp ortance of these postulated events in trp operon regulation, we used s ite-directed mutagenesis to alter the putative elements involved in tr anscriptional and translational control. Using a trpE'-'lacZ reporter, we measured translational yield and specific mRNA levels with various leader constructs, in both mtrB(+) and mtrB strains, during growth in the presence and absence of excess tryptophan, To verify that the alt ered regulatory regions behaved as expected, we carried out in vitro t ranscription assays with the wild-type and altered leader region templ ates and performed oligonucleotide competition assays with an oligonuc leotide complementary to a segment of the transcription terminator. Ou r results establish that binding of TRAP to leader RNA regulates of tr anscription termination in the trp operon over about an 88-fold range and regulates translation of trpE over about a 13-fold range. The role s played by different trp leader RNA segments in mediating transcripti onal and translational regulation are documented by our findings.