E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure

In transcription initiation by Escherichia coli RNA polymerase (RNAP), initial binding to promoter DNA triggers large conformational changes, bending downstream duplex DNA into the RNAP cleft and opening 13 bp to form a short-lived open intermediate (I₂). Subsequent conformational changes increase lifetimes of λP_R and T7A1 open complexes (OCs) by > 10⁵-fold and > 10²-fold, respectively. OC lifetime is a target for regulation. To characterize late conformational changes, we determine effects on OC dissociation kinetics of deletions in RNAP mobile elements σ⁷⁰ region 1.1 (σ_1.1), β′ jaw and β′ sequence insertion 3 (SI3). In very stable OC formed by the wild type WT RNAP with λP_R (RP_O) and by Δσ_1.1 RNAP with λP_R or T7A1, we conclude that downstream duplex DNA is bound to the jaw in an assembly with SI3, and bases - 4 to + 2 of the nontemplate strand discriminator region are stably bound in a positively charged track in the cleft. We deduce that polyanionic σ_1.1 destabilizes OC by competing for binding sites in the cleft and on the jaw with the polyanionic discriminator strand and downstream duplex, respectively. Examples of σ_1.1-destabilized OC are the final T7A1 OC and the λP_R I₃ intermediate OC. Deleting σ_1.1 and either β′ jaw or SI3 equalizes OC lifetimes for λP_R and T7A1. DNA closing rates are similar for both promoters and all RNAP variants. We conclude that late conformational changes that stabilize OC, like early ones that bend the duplex into the cleft, are primary targets of regulation, while the intrinsic DNA opening/closing step is not.