Kinetic Studies and Structural Models of the Association of E. coli σ70 RNA Polymerase with the λPR Promoter: Large Scale Conformational Changes in Forming the Kinetically Significant Intermediates

The kinetics of interaction of Eσ70 RNA polymerase (R) with the λPR 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 (I2, RPo) are single-exponential with first order rate constant βCR. Interpretation of the polymerase concentration dependence of βCR in terms of the three step mechanism of open complex formation yields the equilibrium constant K1 for formation of the first kinetically significant intermediate (I1) and the forward rate constant (k2) for the conformational change converting I1 to the second kinetically significant intermediate I2:R+P⇄K1I1→k2I2.Use of rapid quench mixing allows K1 and k2 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 I1, its relatively small binding constant K1 at 37 °C at this [salt] (∼6×106 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 ka (ΔCao,‡=−1.5(±0.2) kcal K−1), now shown directly to originate largely from formation of I1 (ΔC1o=−1.4(±0.3) kcal K−1), rather than from the formation of I2 as previously proposed. The isomerization I1→I2 exhibits relatively slow kinetics and has a very large temperature-independent Arrhenius activation energy (E2act=34(±2) kcal). This kinetic signature suggests that formation of the transition state (I1–I2)‡ involves large conformational changes dominated by changes in the exposure of polar and/or charged surface to water.