No 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 enzymepromoter 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 cr 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 Al promoter, the phage T5 N25 promoter, and the chimeric promoter T5 N25antiDSR. 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 N25antiDSR 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 Al 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 N25antiDSR 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.

Promoter escape efficiency of E. coli RNA polymerase is guided by both the core promoter and the initial transcribed sequence (ITS). Here, we quantitatively examined the escape properties of 43 random initial sequence variants of the phage T5 N25 promoter. The position for promoter escape on all N25-ITS variants occurred at the +15/+16 juncture, unlike the +11/+12 juncture for the wild type N25. These variants further exhibited a 25-fold difference in escape efficiency. ITS changes favoring promoter escape showed a compositional bias that is unrelated to nucleotide substrate binding affinity for the initial positions. Comparing all variants, the natural N25 promoter emerges as having evolved an ITS optimal for promoter escape, giving a high level of productive synthesis after undergoing the shortest abortive program. We supplemented GreB to transcription reactions to better understand abortive initiation and promoter escape in vivo. GreB supplementation elevated productive RNA synthesis 2-5-fold by altering the abortive RNA pattern, decreasing the abundance of the medium (6-10 nt) to long (11-15 nt) abortive RNAs without changing the levels of short (2-5 nt) and very long abortive RNAs (16-20 nt). The GreB-refractive nature of short abortive RNA production may reflect a minimum length requirement of 4-5 bp of the RNA-DNA hybrid for maintaining the stability of initial or backtracked complexes. That the very long abortive RNAs are unaffected by GreB suggests that they are unlikely to be products of polymerase backtracking. How the ITS might influence the course of early transcription is discussed within the structural context of an initial transcribing complex.

Abortive initiation and promoter escape are two principal biochemical reactions occurring in the latter stage of transcript initiation. We have analyzed the influences of individual DNA elements within the promoter recognition region (PRR) on these reactions by measuring the quantitative initiation parameters that describe abortive initiation and promoter escape; these parameters are the abortive rate, the productive rate, the abortive:productive ratio, the abortive probability, and the maximum size of abortive transcripts. Changes in the individual DNA elements within the PRR can have a substantial effect on each of these parameters. The discriminator region and the -10 element primarily influence the abortive probability at positions 2-5 and 6-10, respectively, while the -10 and -35 conserved hexamers and the spacer region affect the abortive probability at positions 11-15. Surprisingly, transcription of a consensus promoter invariably gives a higher abortive yield, a higher abortive probability, a longer abortive ladder, and a lower productive rate than promoter variants carrying even a single deviation in the consensus hexamers. These results suggest that strong RNA polymerase-PRR interactions stall the polymerase at the promoter, thereby reducing the rate of promoter escape and consequently enhancing the extent of abortive initiation.