Rapid Quench Mixing to Quantify Kinetics of Steps in Association of Escherichia coli RNA Polymerase with Promoter DNA

Abstract

This chapter explores the kinetic–mechanistic studies that have characterized various steps in the process of forming the transcriptionally competent open complex between RNA polymerase (R) and promoter DNA (P). It discusses the rapid quench mixing of radiolabeled λP R promoter DNA with E. coli RNA polymerase in a commercially available apparatus providing an accurate and efficient method of investigating the kinetics of association, and subsequent conformational changes involved in forming long-lived complexes. Promoter binding as a function of time is assayed by nitrocellulose filter binding after quenching with a competitor. Under all conditions examined, the kinetics of formation of competitor-resistant complexes at the λPR promoter are single exponential with first-order rate constant β CR . Interpretation of the polymerase concentration dependence of β CR in terms of the three-step mechanism of open complex formation yields the equilibrium constant K 1 for formation of the first kinetically significant intermediate (I 1 ) and the forward rate constant (k 2 ) for the conformational change that converts I1 to the second kinetically significant intermediate I 2. This method should be applicable to kinetic studies of polymerase–promoter interactions and thus should extend biophysical characterizations of transcription initiation.