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 λPR 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 (σ1.1), β′ jaw and β′ 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 β′ 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.
Bibliographical noteFunding Information:
We thank Dr. Ruth Saecker for her insights into and discussions of RNAP and the conformational changes in transcription initiation that led to this research. We thank Dr. Ran Furman and Dr. Caroline Davis for initial experiments. We thank Prof. Richard Gourse, Prof. Robert Landick and their laboratories for helpful discussions. This work was supported by the National Institutes of Health ( GM103061 to M.T.R. and GM067153 to I.A.).
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- Abbreviations OC open complex
- CRE core recognition element
- DME downstream mobile element
- NT nontemplate
- RNAP RNA polymerase
- TB Tris buffer