Mechanisms of Very Long Abortive Transcript Release during Promoter Escape

A phage T5 N25 promoter variant, DG203, undergoes the escape transition at the +16 to +19 positions after transcription initiation. By specifically examining the abortive activity of the initial transcribing complex at position +19 (ITC19), we observe the production of both GreB-sensitive and GreB-resistant VLAT19. This suggests that ITC19, which is perched on the brink of escape, is highly unstable and can achieve stabilization through either backtracking or forward translocation. Of the forward-tracked fraction, only a small percentage escapes normally (followed by stepwise elongation) to produce full-length RNA; the rest presumably hypertranslocates to release GreB-resistant VLATs. VLAT formation is dependent not only on consensus −35/–10 promoters with 17 bp spacing but also on sequence characteristics of the spacer DNA. Analysis of DG203 promoter variants containing different spacer sequences reveals that AT-rich spacers intrinsically elevate the level of VLAT formation. The AT-rich spacer of DG203 j...