Temperature dependence of the rate constants of the Escherichia coli RNA polymerase-lambda PR promoter interaction. Assignment of the kinetic steps corresponding to protein conformational change and DNA opening.

Abstract The kinetics of formation and of dissociation of open complexes (RP O ) between Escherichia coli RNA polymerase (R) and the λP R promoter (P) have been studied as a function of temperature in the physiological range using the nitrocellulose filter binding assay. The kinetic data provide further evidence for the mechanism R + P ⇋ I 1 ⇋ I 2 ⇋ RP O , where I 1 and I 2 are kinetically distinguishable intermediate complexes at this promoter which do not accumulate under the reaction conditions investigated. The overall second-order association rate constant ( k a ) increases dramatically with increasing temperature, yielding a temperature-dependent activation energy in the range 20 kcal (near 37 °C) to 40 kcal (near 13 °C) (1 kcal = 4.184 kJ). Both isomerization steps (I 1 → I 2 and I 2 → RP 0 ) appear to be highly temperature dependent. Except at low temperatures ( 1 → I 2 , which we attribute to a conformational change in the polymerase with a large negative δC p o value, is rate-limiting at the reactant concentrations investigated and hence makes the dominant contribution to the apparent activation energy of the pseudo first-order association reaction. The subsequent step I 2 → RP O , which we attribute to DNA melting, has a higher activation energy (in excess of 100 kcal) but only becomes rate-limiting at low temperature ( 1 appears to be in equilibrium on the time-scale of the isomerization reactions under all conditions investigated; the equilibrium constant for this step is not a strong function of temperature and is approximately 10 7 m −1 under the standard ionic conditions of the assay (40 m m -Tris HCl (pH 8.0), 10 m m -MgCl 2 , 0.12 m -KCl). The activation energy of the dissociation reaction becomes increasingly negative at low temperatures, ranging from approximately −9 kcal near 37 °C to −30 kcal near 13 °C. Thermodynamic (van't Hoff) enthalpies ΔH o of open complex formation consequently are large and temperature-dependent, increasing from approximately 29 to 70 kcal as the temperature is reduced from 37 to 13 °C. The corresponding ΔC p o value is approximately − 2.4 kcal/deg. We propose that this large negative ΔC p o value arises primarily from the burial of hydrophobic surface in the conformational change (I 1 ⇋ I 2 ) in RNA polymerase in the key second step of the mechanism. The three-step mechanism for open complex formation suggests a division of promoters into two classes: sequential R + P → k 1 I 1 → k 2 + I 2 → k 3 → RP 0 and rapid binding equilibrium R + P ⇋ K 1 I 1 → K 2 I 2 → K 3 RP 0 . The pseudo first-order time constants τ obs for these two classes ( τ obs = ( k a R T ) −1 + k 2 −1 + k 3 −1 ) differ only in the interpretation of k a , and hence in their behavior at low polymerase concentration ( k a = k 1 (sequential); k a = K 1 k 2 (rapid equilibrium)). At high polymerase concentration, values of τ obs for both classes are determined by the slower of the two isomerization steps, which in general respond differently to temperature and other regulatory variables. Consequences of this mechanistic decomposition for the behavior of various promoters in vivo and in vitro are explored.