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

ABSTRACT: 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 ?PR 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 ?(70) region 1.1 (?1.1), ?' jaw and ?' sequence insertion 3 (SI3). In very stable OC formed by the wild type WT RNAP with ?PR (RPO) and by ??1.1 RNAP with ?PR 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 ?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 ?1.1-destabilized OC are the final T7A1 OC and the ?PR I3 intermediate OC. Deleting ?1.1 and either ?' jaw or SI3 equalizes OC lifetimes for ?PR 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.

SUBMITTER: Ruff EF 

PROVIDER: S-EPMC4520765 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature