Kinetics of promoter search by Escherichia coli RNA polymerase. Effects of monovalent and divalent cations and temperature

The rapid mixing/photocross-linking technique developed in our laboratory has been employed in the study of the mechanism of promoter binding by Escherichia coli RNA polymerase (RPase). We have previously reported on the quantitation of the one-dimensional diffusion coefficient (D₁) for RPase along the DNA template (Singer, P. T., and Wu, C.-W. (1987) J. Biol. Chem. 262, 14178-14189). In this paper, we describe the effect of salt concentration and temperature on the kinetics of promoter search by RPase using plasmid pAR1319 DNA, which contains the A2 early promoter from bacteriophage T7, as template. Over a range of KCl concentrations from 25 to 200 mM, the apparent bimolecular rate constant (k(a)) for the association of RPase with the A2 promoter on this DNA template varied approximately 2-fold, achieving a maximal value between 100 and 125 mM KCl. More significantly, the transient distribution of RPase among nonspecific DNA binding sites changed markedly as a function of salt concentration, indicative of gross changes in the average number of base pairs covered by sliding during a nonspecific lifetime. Using the mathematical treatment outlined in our earlier report, the nonspecific dissociation rate constant (k(off)) was calculated from the binding curves for the nonspecific as well as promoter-containing DNA. The observed variations in k(a) as a function of monovalent cation concentration ([M⁺]) were due primarily to changes in k(off), as D₁ was found to be essentially independent of [M⁺]. Interestingly, D₁ decreased by one-third as the concentration of magnesium was lowered from 10 to 1 mM. In addition, the depence of k(off) (and consequently the nonspecific equilibrium association constant, K(eq)) on [M⁺] agreed qualitatively with the results of deHaseth et al. (deHaseth, P. L., Lohman, T. M., Burgess, R. R., and Record, M. T., Jr. (1977) Biochemistry 17, 1612-1622), though we consistently measure a weaker K(eq). The association rate constant was also measured between 4 and 37°C, and was found to vary ~ 2-fold over that range. An activation energy for the bimolecular association of RPase to the A2 promoter was calculated to be 2.2 ± 0.4 kcal/mol, while the activation energy for one-dimensional diffusion was 4.7 ± 0.8 kcal/mol.