Escherichia coli transcript cleavage factors GreA and GreB stimulate promoter escape and gene expression in vivo and in vitro

Abstract

The process of RNA chain initiation by RNA polymerases plays a central role in the regulation of transcription. In this complex phase of transcription, short oligomers are synthesized and released from the enzyme- promoter complex in a reaction termed abortive initiation. The polymerase undergoes many cycles of abortive initiation prior to completion of the initiation process, which is signaled by the translocation of the enzyme away from the promoter, release of θ factor, and formation of an elongation complex in which the RNA is stably bound. We have studied the parameters that affect escape from the promoter by Escherichia coli RNA polymerase for the phage T7 A1 promoter, the phage T5 N25 promoter, and the chimeric promoter T5 N25(antiDSR). The latter site contains a synthetic initial transcribed region that reduces its ability to synthesize RNA both in vivo and in vitro. Clearance from T5 N25(antiDSR) can be stimulated up to 10-fold in vitro by addition of the E. coli transcript cleavage factor GreA or GreB, but these factors have little effect on transcription from the normal T7 A1 or T5 N25 promoters. Using an E. coli strain lacking GreA and GreB, we were also able to show stimulation of transcription by the Gre factors from the T5 N25(antiDSR) promoter in vivo. The stimulation of RNA chain initiation by Gre factors, together with their known biochemical properties in the transcription elongation reaction, suggests some specific models for steps in the transcription initiation reaction.