Late Steps in the Formation of E. coli RNA Polymerase—λPR Promoter Open Complexes: Characterization of Conformational Changes by Rapid [Perturbant] Upshift Experiments

Abstract

The formation of the transcriptionally competent open complex (RPo) by Escherichia coli RNA polymerase at the λPR promoter involves at least three steps and two kinetically significant intermediates (I1 and I2). Understanding the sequence of conformational changes (rearrangements in the jaws of RNA polymerase, DNA opening) that occur in the conversion of I1 to RPo requires: (1) dissecting the rate constant kd for the dissociation of RPo into contributions from individual steps and (2) isolating and characterizing I2. To deconvolute kd, we develop experiments involving rapid upshifts to elevated concentrations of RPo-destabilizing solutes (“perturbants”: urea and KCl) to create a burst in the population of I2. At high concentrations of either perturbant, kd approaches the same [perturbant]-independent value, interpreted as the elementary rate constant k−2 for I2→I1. The large effects of [urea] and [salt] on K3 (the equilibrium constant for I2⇄RPo) indicate that a large-scale folding transition in polymerase occurs and a new interface with the DNA forms late in the mechanism. We deduce that I2 at the λPR promoter is always unstable relative to RPo, even at 0 °C, explaining previous difficulties in detecting it by using temperature downshifts. The division of the large positive enthalpy change between the late steps of the mechanism suggests that an additional unstable intermediate (I3) may exist between I2 and RPo.