RNA polymerase pausing and transcript release at the lambda tR1 terminator in vitro.

Abstract

Transcription of the lambda rightward operon encounters multiple tandem p-dependent termination sites at a region known as tR1, yielding the mRNA for the lytic repressor cro. We show that in the absence of p factor, RNA polymerase pauses at each of three p-dependent termination sites (I, II, and III) at tR1 during in vitro transcription. We demonstrate that p factor itself does not affect transcriptional pausing at these sites. Relative occupancies of the pause sites during initial transcription have been measured. The lambda cin-1cnc-1 mutations, which abolish or vastly reduce termination at site II of tR1, nevertheless elicit transcriptional pausing at that site. The NusA protein (L factor) prolongs these transcriptional pauses to varying degrees. Transcriptional pausing at termination site II is dramatically enhanced by NusA in lambda wild type, but not in lambda cin-1cnc-1. NusA enhances pausing at site I but inhibits termination at that site, suggesting that it may also affect transcript release. We show that NusA does alter the kinetics of transcript release from isolated ternary complexes. We suggest that the ability of the NusA protein to increase or decrease termination efficiencies at various sites is attributed to its ability to modulate transcriptional pausing as well as transcript release.