Expression of the Bacillus subtilis trpEDCFBA operon is influenced by translational coupling and Rho termination factor

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and transla tional control mechanisms. Both mechanisms require binding of tryptophan-ac tivated TRAP to 11 (G/U)AG repeats in the tip leader transcript. trpE trans lational control involves formation of a TRAP-dependent RNA structure that sequesters the trpE Shine-Dalgarno (SD) sequence (the SD blocking hairpin). By comparing expression levels from trpE'-'lacZ translational fusions cont rolled by the wild-type leader or by a leader that cannot form the SD block ing hairpin, we found that translational control requires a tryptophan conc entration higher than that required for transcription attenuation. We also found that inhibition of trpE translation by the SD blocking hairpin does n ot alter the stability of the downstream message. Since the coding sequence s for trpE and trpD overlap by 29 nucleotides, we examined expression level s from trpED'-'lacZ translational fusions to determine if these two genes a re translationally coupled. We found that introduction of a UAA stop codon in trpE resulted in a substantial reduction in expression. Since expression was partially restored in the presence of a tRNA suppressor, our results i ndicate that trpE and trpD are translationally coupled. We determined that the coupling mechanism is TRAP independent and that formation of the SD blo cking hairpin regulates trpD translation via translational coupling. We als o constructed a rho mutation to investigate the role of Rho-dependent termi nation in trp operon expression. We found that TRAP-dependent formation of the SD blocking hairpin allows Rho access to the nascent transcript, causin g transcriptional polarity.