Characterization of TRAP-mediated regulation of the B. subtilis trp operon using in vitro transcription and transcriptional reporter fusions in vivo

Abstract

In Bacillus subtilis, transcription of the tryptophan biosynthetic operon is regulated by an attenuation mechanism involving two alternative RNA secondary structures in the 5′ leader region upstream of the structural genes. Regulation is accomplished, at least in part, by controlling which RNA structure forms during transcription of the operon. When intracellular tryptophan levels are high, the trp R NA-binding a ttenuation p rotein (TRAP) binds to the nascent trp mRNA to promote formation of a transcription terminator structure so as to induce transcription termination prior to the structural genes. In limiting tryptophan, TRAP does not bind, the alternative antiterminator RNA structure forms, and the operon is transcribed. Several in vitro and in vivo assays have been utilized to study TRAP-mediated regulation of both transcription and translation. Here, we describe using in vitro transcription attenuation assays and in vivo trp-lacZ fusions to examine TRAP-mediated regulation of the trp genes.