Pausing of RNA polymerase during in vitro transcription of the tryptophan operon leader region

Abstract

RNA polymerase molecules pause at a single site during in vitro transcription of the tryptophan (trp) operon leader region. Pausing was observed when DNA templates derived from Escherichia coli. Salmonella typhimurium, and Klebsiella aerogenes were used. Fingerprint analyses showed that the major RNA species produced by the transcriptional pause is 91 nucleotides long. A minor RNA species 90 nucleotides long was also detected. Single-round transcription experiments were used to study the kinetics of pausing. Time course, pulse-chase, and delayed-labeling experiments suggest that every RNA polymerase molecule transcribing the trp leader region pauses. A suboptimal ribonucleoside triphosphate concentrations, the half-life of paused-leader RNA was approximately 3 min at 22 degrees C and 0.7 min at 37 degrees C. At near-optimal ribonucleoside triphosphate concentrations, the half-time of the paused species dropped to about 0.3 min at 22 degrees C. The appearance and half-life of the paused species were unaffected by salt concentration, rho factor, guanosine 3'-5'-bis(diphosphate), or point mutations in the trp attenuator region. It is postulated that transcriptional pausing may play a role in maintaining the synchronization of transcription and translation that is vital in the control of transcription termination at the trp operon attenuator.