The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis regulates translation initiation of ycbK, a gene encoding a putative efflux protein, by blocking ribosome binding

Expression of the Bacillus subtilis tryptophan biosynthetic genes trpEDCFBA and trpG, as well as a putative tryptophan transport gene (trpP), are regulated in response to tryptophan by the trp RNA-binding attenuation protein (TRAP). TRAP regulates expression of these genes by transcription attenuation and translation control mechanisms. Here we show that TRAP also regulates translation of ycbK, a gene that encodes a protein with similarities to known efflux proteins. As a likely TRAP-binding site consisting of 11 NAG repeats overlaps the ycbK translation initiation region, experiments were carried out to determine whether TRAP regulates translation of ycbK. TRAP was observed to regulate expression of a ycbK'-'lacZ translational fusion 20-fold in response to tryptophan. Binding studies indicated that TRAP binds to the ycbK transcript with high affinity and specificity. Footprint studies revealed that the central seven triplet repeats were protected by bound TRAP, while toeprint results suggest that nine triplet repeats contribute to TRAP binding. Additional toeprint and in vitro translation analyses demonstrated that bound TRAP regulates YcbK synthesis by blocking ribosome binding. We also identified two dipeptide coding minigenes between the Shine-Dalgarno sequence and start codon of ycbK. Expression of one of the minigenes modestly interfered with translation of ycbK.