Kinetic studies and structural models of the association of E. coli σ⁷⁰ RNA polymerase with the λP_R promoter: Large scale conformational changes in forming the kinetically significant intermediates

The kinetics of interaction of Eσ⁷⁰ 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 (I₂, 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 Κ₁ for formation of the first kinetically significant intermediate (I₁) and the forward rate constant (κ₂) for the conformational change converting I₁ to the second kinetically significant intermediate I₂:R + P ⇄^Κ1 I₁ →^k2 I₂. Use of rapid quench mixing allows Κ₁ and κ₂ 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 Κ₁ 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 κ_a (ΔCo_a/^o,‡ = -1.5(±0.2) kcal Κ^-1), now shown directly to originate largely a from formation of I₁ (ΔC₁/^o = -1.4(±0.3) kcal Κ^-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 (E₂/^act = 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. Structural and biochemical data lead to the following hypotheses to interpret these results. We propose that formation of I₁ involves coupled folding of unstructured regions of polymerase (β, β′ and σ⁷⁰) and bending of promoter DNA (in the -10 region). We propose that interactions with region 2 of σ⁷⁰ 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 I₁. 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.