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Crosslinking very long abortive transcripts (VLATs) to E. coli RNA polymerase: an approach to elucidate forward hyper‐translocation

On a typically strong E. coli promoter, as nascent RNA grows to 8–10 nt—accompanied by scrunching 6–8 bp of the enlarged transcription bubble—the initial DNA‐RNAP contacts disrupt, upstream bubble rewinds, promoter escape occurs, and the elongation phase ensues. On some unique promoters, escape is accomplished only when the nascent RNA has grown to 16–19 nt in length. Such a promoter on the cusp of escape contains extremely high stress energy, which when utilized for upstream bubble rewinding and allowing promoter escape, can generate so much force as to propel the RNAP forward by several bases (Chander et al. 2007 Biochemistry 46, 12687). We call this process forward hyper‐translocation, during which the nascent RNA 3’‐OH is moved upstream into the RNA exit channel, out of register with the active site. The RNA molecule cannot be further elongated and is released as a VLAT. This mechanism can account for the GreB‐resistant nature of VLAT synthesis. To prove the occurrence of forward hyper‐translocation, I incorporated a 4‐thiouridine (S4U) residue at the 3′ end of a 19‐nt VLAT and conducted UV crosslinking experiments followed by SDS‐PAGE analysis. Our results showed UV‐, RNAP‐, and S4U‐dependent crosslinking of the VLAT to the β/β′ subunits. Using mass spectrometry, we are determining the location of the crosslink, and whether it coincides with the RNA exit channel. Supported by NSF‐RUI grant (MCB 0841452) to L. M. Hsu