An alternate mechanism of abortive release marked by the formation of very long abortive transcripts

The E sigma(70)-dependent N25 promoter is rate-limited at promoter escape. Here, RNA polymerase repeatedly initiates and aborts transcription, giving rise to a ladder of short RNAs 2-11 nucleotides long. Certain mutations in the initial transcribed sequence (ITS) of N25 lengthen the abortive initiation program, resulting in the release of very long abortive transcripts (VLATs) 16-19 nucleotides long. This phenomenon is completely dependent on sequences within the first 20 bases of the ITS since altering sequences downstream of +20 has no effect on their formation. VLAT formation also requires strong interactions between RNA polymerase and the promoter. Mutations that change the -35 and -10 hexamers and the intervening 17 base pair spacer away from consensus decrease the probability of aborting at positions +16 to +19. An unusual characteristic of the VLATs is their undiminished levels in the presence of GreB, which rescues abortive RNAs (:! 15 nucleotides) associated with backtracked initial transcribing complexes. This suggests that VLATs are produced via a mechanism distinct from backtracking, which we propose entails polymerase molecules hyper forward translocating during the promoter escape transition. We discuss how certain features in the ITS, when combined with the N25 promoter, may lead to hyper forward translocation and abortive release at VLAT positions.