Fluorescence Resonance Energy Transfer Characterization of DNA Wrapping in Closed and Open Escherichia coli RNA Polymerase-λP(R) Promoter Complexes.

Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40-50 bp of DNA immediately upstream of the -35 region. Kinetic studies demonstrated that the presence of upstream DNA facilitates bending and entry of the downstream duplex (to +20) into the active site cleft to form an advanced closed complex (CC), prior to melting of ∼13 bp (-11 to +2), including the transcription start site (+1). Atomic force microscopy and footprinting revealed that the stable open complex (OC) is also highly wrapped (-60 to +20). To test the proposed bent-wrapped model of duplex DNA in an advanced RNAP-λP(R) CC and compare wrapping in the CC and OC, we use fluorescence resonance energy transfer (FRET) between cyanine dyes at far-upstream (-100) and downstream (+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies are observed for the CC (0.30 ± 0.07) and the OC (0.32 ± 0.11) for both probe orientations. Fluorescence enhancements at +14 are observed in the single-dye-labeled CC and OC. These results demonstrate that upstream DNA is extensively wrapped and the start site region is bent into the cleft in the advanced CC, reducing the distance between positions -100 and +14 on promoter DNA from >300 to <100 Å. The proximity of upstream DNA to the downstream cleft in the advanced CC is consistent with the proposed mechanism for facilitation of OC formation by upstream DNA.

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 13bp to form a short-lived open intermediate (I2). Subsequent conformational changes increase lifetimes of λPR and T7A1 open complexes (OCs) by >10(5)-fold and >10(2)-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 σ(70) region 1.1 (σ1.1), ß' jaw and ß' sequence insertion 3 (SI3). In very stable OC formed by the wild type WT RNAP with λPR (RPO) and by Δσ1.1 RNAP with λPR 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 σ1.1 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 σ1.1-destabilized OC are the final T7A1 OC and the λPR I3 intermediate OC. Deleting σ1.1 and either ß' jaw or SI3 equalizes OC lifetimes for λPR 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.