Differential Effects of Physiological Pyrophosphate on the Kinetics of Productive and Abortive Transcription Initiation

Abstract

Transcription initiation is the first and most highly regulated step of gene expression, and a biophysical characterization of its mechanism is needed. Previous studies found two, roughly equal populations of DNA‐RNA open complexes (OCs) in transcription initiation: productive OC that escape from the promoter to make full length (FL) RNA and nonproductive OC that stall after synthesis of a short RNA, slowly release that RNA, and reinitiate (abortive initiation). The kinetics and mechanism of productive transcription initiation by E. coli RNA polymerase (RNAP) at the λPR promoter were recently determined without added pyrophosphate (PPi).1 Because each polymerization step of transcription releases PPi and total PPi concentrations in the cell are in the low mM range, it is important to determine the effects (e.g. product inhibition) of PPi on the catalytic and translocation steps of initiation, and compare with recent results for PPi effects on elongation.2 In the research reported here, we extend existing quantitative 32P‐detected initiation kinetic assays to physiological concentrations of PPi and assess PPi effects on both the amounts and rates of the steps of productive and abortive initiation. Using phosphorimager analysis of gel separations and one‐dimensional thin layer chromatography, we study single‐round initiation of FL RNA synthesis at RNAP‐λPR open complexes under conditions where overall forward rate constants for each step of NTP incorporation in hybrid extension up to and beyond the escape point (11bp for initiation from λPR promoter) were previously determined.1 This will allow determination of PPi effects on each step, and potentially a dissection into effects on translocation and catalytic steps. Results to date show distinct PPi effects on productive and nonproductive initiation. The level of FL RNA synthesis by productive complexes is significantly reduced, but the level of abortive initiation is increased, indicating that previously‐productive OC are stalled by PPi. An understanding of PPi effects on productive and abortive initiation is critical to future assessments of the regulation and fidelity of bacterial transcription initiation in vivo, and the in vivo significance of short abortive RNAs as regulators.