Real-Time Initial Transcription by a Multisubunit RNA Polymerase

Abstract

Abortive initiation, the repetitive synthesis of short RNA transcripts before promoter escape and elongation, is a highly dynamic process that can constitute the rate-limiting step for many gene promoters. Previous studies using single-molecule Förster Resonance Energy Transfer (smFRET) demonstrated that abortive initiation occurs via a DNA-scrunching mechanism mediated by RNA polymerase. While these studies, along with biochemical work, have yielded insight into abortive initiation, no single-molecule method has been able to directly capture RNA synthesis/release, or the associated conformational changes of the transcription complex. Here, we descibe a highly sensitive smFRET assay that, for the first time, monitors transcription bubble expansion and compaction during abortive RNA synthesis and release in real-time; we also performed similar analysis using an assay that reports on the movement of downstream DNA toward the RNA polymerase main channel. Using single-molecule time trajectories, we have observed multiple cycles (up to 50) of RNA synthesis by single RNA polymerase molecules and we used them to obtain the rates of RNA synthesis and release, as well as to study the dependence of the rates on nucleotide concentration and promoter sequence. Our initial results revealed that, surprisingly, the rate of nucleotide addition in initial transcription is 5-10 fold faster than elongation; moreover, we observed large heterogeneity between different RNA polymerase molecules with regards to specific steps in initial transcription; such heterogeneity may contribute to the noise associated with gene expression.