Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the λPR promoter

Abstract

The kinetics of formation and dissociation of specific (open) complexes between active Escherichia coli RNA polyrmerase holoenzyme (RNAP) and the λP R promoter have been studied by selective nitrocellulose filter binding assays at two temperatures (25 °C, 37 °C) and over a range of ionic conditions. Competition with a polyanion (heparin) or stabilization of open promoter complexes at P R by incubation with specific combinations of nucleoside triphosphates was employed to obtain selectivity in the filter assay. This study provides a useful example of how information about mechanism may be obtained from the quantitative analysis of the effects of salt concentration and temperature on the rate constants of a protein-DNA interaction. The association reaction between RNAP and λP R was investigated under ionic conditions where the process is essentially irreversible, and under pseudo first-order conditions of excess polymerase. The pseudo first-order rate constant is directly proportional to the concentration of active polymerase over the entire range investigated (2 to 10 nM) at both 25 °C and 37 °C. within experimental uncertainty. Second-order association rate constants ( k a), calculated from these data at standard ionic conditions (0.12 m-KCl, 0.01 m-MgCl 2, 0.04 m-Tris (pH 8)), were strongly temperature-dependent: k a = (2.6 ± 0.4) × 10 6 m −1 s −1 at 37 °C and k a = (7.2 ± 1.4) × 10 5 m −1 s −1 at 25 °C, corresponding to an activation energy of the association reaction of approximately 20 ± 5 kcal. In addition, k a decreases strongly with increasing KCl concentration, corresponding to the net release of the thermodynamic equivalent of at least nine monovalent ions prior to or during the rate-limiting step of the association reaction. This strong dependence of k a on the ionic environment suggests that inorganic cations should be considered as possible regulators of in vivo transcription initiation. Dissociation rate constants ( k d) were also measured under irreversible reaction conditions. At the standard ionic conditions, k d = (2.2 ± 0.3) × 10 −5 s −1 at 37 °C and k d = (4.0 ± 0.4) × 10 −5 s −1 at 25 °C. The increase in k d with decreasing temperature corresponds to a negative activation energy of dissociation (−9 ± 4 kcal). In addition. k d increases with increasing KCl concentration, corresponding to the net uptake of the thermodynamic equivalent of at least six monovalent ions in or prior to the rate-limiting step of the dissociation reaction. Equilibrium constants K obs RP = k a k d for the association of RNA polymerase with the P R promoter are also strong functions of temperature and ionic conditions. At 37 °C in the standard buffer, K obs RP = (1.2 ± 0.3) × 10 11 m −1. The van't Hoff enthalpy ΔH 0 of the process is estimated to be 29 ± 9 kcal. The thermodynamic equivalent of at least 15 monovalent ions is released in the overall process of association. The entropic contribution from release of these ions provides much of the thermodynamic driving force for the binding reaction under the ionic conditions investigated. We argue on the basis of the magnitude of k a and the dependences of k a and k d on temperature and salt concentration that a minimum reaction mechanism for the interaction of RNA polymerase (R) with the λP R promoter (P) to form an open complex (RP o) must be: R + P ↔ I 1 ↔ I 2 ↔ RP o. where I 1 and I 2 are intermediates which do not accumulate under the reaction conditions investigated and where the rate-limiting steps, under the conditions investigated, are the interconversions of the transient intermediates I 1 and I 2, which presumably correspond to conformationally distinct forms of an intermediate (closed) promoter complex. The kinetic constants for the individual steps in the association reaction differ significantly from those previously obtained for P R from the abortive initiation assay (Hawley & McClure, 1980), although the overall association rate constants ( k a) measured by the two assays are comparable. The general applicability of the above mechanism to RNA polymerase-promoter interactions is discussed.