|dc.description.abstract||Transcription is one of the highly regulated steps in gene expression and comprises three phases – initiation, elongation and termination. In prokaryotes, the initiation phase is further divided into two steps: (1) promoter binding and activation; and (2) abortive initiation and promoter escape. Our wild-type promoter of interest, T5 phage N25, is a prototypical strong E. coli RNA polymerase σ70 promoter that produces abundant transcripts. However, due to its consensus-like promoter elements, the transcription initiation is rate-limited at the promoter escape step, leading to extensive abortive initiation. Studies have shown that the position of promoter escape depends not only on the promoter-polymerase affinity but also on the initial transcribed sequence (ITS) (Kammerer et al., 1986; Hsu et al., 2006). While N25 aborts to +11 and escapes at +12, its ITS variants, such as N25anti and DG203, abort to +15 and +19 and escape at +16 and +20, respectively (Hsu et al., 2006).
Studies with the transcription factor GreB showed that all abortive transcripts ≤ 15 nucleotides (nt) arise from RNA polymerase (RNAP) backtracking and those longer than 4 nt can be rescued (Hsu et al., 1995). “Rescue” involves GreB, bound in the RNAP secondary channel, stimulating the hydrolytic activity of RNAP to cleave the backtracked nascent RNA at the active site, aligning a new 3′-OH group such that the 5′-nascent RNA can be further elongated (Opalka et al., 2003). However, on the DG203 promoter, it was discovered that a fraction of the very long abortive transcripts (VLATs) of 16-19 nt in length that are GreB-resistant. Consequently, it was proposed that the GreB-resistant VLATs emerge from a different mechanism called RNAP hyper-forward translocation: Upon reaching the promoter escape transition stage, a fraction of the RNAP is propelled forward by more than one nucleotide, causing the 3'-end of nascent RNA to move upstream past the active center and become lodged in the RNA exit channel instead. Thus inactivated, the RNA polymerase complex subsequently releases the nascent RNA as abortive transcripts (Chander et al., 2007).
In the current study, the proposition of GreB-resistant VLAT formation by RNAP hyper-forward translocation was tested by placing a mutant EcoRI protein roadblock at various locations in the downstream stretches of DG203 promoter and performing in vitro transcription to examine the progress of RNAP. The results of “roadblock transcription” analysis gave the following conclusions: 1. VLATs are indeed products of RNAP forward movement, and they are produced during the promoter escape transition on the DG203 promoter; 2. We observed a spatial requirement of at least 2 bp, but optimally 4-5 bp, for RNAP to undergo hyper forward translocation to produce the GreB-resistant VLATs.
Moreover, it was postulated that the energy that propels RNAP forward by more than one nucleotide comes from DNA scrunching (Revyakin et al., 2006; Chander et al., 2007). If true, the initial transcribing complex of DG203 on the cusp of escape must have scrunched in 19 bp of DNA, greatly expanding the transcription bubble from its normal 13-14 bp size. To test this prediction, permanganate footprinting approach was used to characterize the sizes of the initial transcribing complex (ITC) bubbles. For comparison, a variant of the DG203 promoter called SPfullcon was further created. It differs in sequence from DG203 only in the 17-bp spacer region connecting the -35 and -10 elements. SPfullcon shows the same position of escape as DG203, but its level of full-length transcripts is greatly diminished (Chander and Hsu, manuscript in submission), allowing us to exclusively capture the melted DNA region during initiation and scrunching. Using four different promoters − N25, N25anti, DG203 and SPfullcon − the sizes of the open complex and transcribing complex bubbles were characterized and compared. The results indicated that the VLAT-producing ITC bubbles were much larger; bubble expansion to the +20 position could be detected. This observation suggested a higher degree of DNA scrunching in the DG203 and SPfullcon promoters which, in turn, propelled the RNAP forward by more than one nucleotide during the escape transition, giving rise to the formation of GreB-resistant VLATs.||en_US