Kinetic Studies and Structural Models of the Association of E. coli σ 70 RNA Polymerase with the λP R Promoter: Large Scale Conformational Changes in Forming the Kinetically Significant Intermediates

Abstract

The kinetics of interaction of Eσ 70 RNA polymerase (R) with the λP R promoter (P) were investigated by filter binding over a broad range of temperatures (7.3–42 °C) and concentrations of RNA polymerase (1–123 nM) in large excess over promoter DNA. Under all conditions examined, the kinetics of formation of competitor-resistant complexes (I 2, RP o) 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 converting I 1 to the second kinetically significant intermediate I 2: R+ P ⇄ K 1 I 1 → k 2 I 2. Use of rapid quench mixing allows K 1 and k 2 to be individually determined over the entire temperature range investigated, previously not possible at this promoter using manual mixing. Given the large (>60 bp) interface formed in I 1, its relatively small binding constant K 1 at 37 °C at this [salt] (∼6×10 6 M −1) strongly argues that binding free energy is used to drive large-scale structural changes in polymerase and/or promoter DNA or other coupled processes. Evidence for coupling of protein folding is provided by the large and negative activation heat capacity of k a ( ΔC a o,‡ =−1.5(±0.2) kcal K −1), now shown directly to originate largely from formation of I 1 ( ΔC 1 o =−1.4(±0.3) kcal K −1), rather than from the formation of I 2 as previously proposed. The isomerization I 1→I 2 exhibits relatively slow kinetics and has a very large temperature-independent Arrhenius activation energy (E 2 act =34(±2) kcal). This kinetic signature suggests that formation of the transition state (I 1–I 2) ‡ involves large conformational changes dominated by changes in the exposure of polar and/or charged surface to water. Structural and biochemical data lead to the following hypotheses to interpret these results. We propose that formation of I 1 involves coupled folding of unstructured regions of polymerase (β, β′ and σ 70) and bending of promoter DNA (in the −10 region). We propose that interactions with region 2 of σ 70 and possibly domain 1 of β induce a kink at the −11/−12 base pairs of the λP R promoter which places the downstream DNA (−5 to +20) in the jaws of the β and β′ subunits of polymerase in I 1. These early interactions of β and β′ with the DNA downstream of position −5 trigger jaw closing (with coupled folding) and subsequent steps of DNA opening.