General method of analysis of kinetic equations for multistep reversible mechanisms in the single-exponential regime:: Application to kinetics of open complex formation between Eσ70 RNA polymerase and λPR promoter DNA

A novel analytical method based on the exact solution of equations of kinetics of unbranched first- and pseudofirst-order mechanisms is developed for application to the process of E sigma(70) RNA polymerase (R)-lambda P-R promoter (P) open complex formation, which is described by the minimal three-step mechanism with two kinetically significant intermediates (I-1, I-2), [GRAPHICS] where the final product is an open complex RPo. The kinetics of reversible and irreversible association (pseudofirst order, [R] much greater than [P]) to form long-lived complexes (RPo and I-2) and the kinetics of dissociation of long-lived complexes both exhibit single exponential behavior. In this situation, the analytical method provides explicit expressions relating observed rate constants to the microscopic rate constants of mechanism steps without use of rapid equilibrium or steady-state approximations, and thereby provides a basis for interpreting the composite rate constants of association (k(a)), isomerization (k(i)), and dissociation (k(d)) obtained from experiment for this or any other sequential mechanism of any number of steps. in subsequent papers, we apply this formalism to analyze kinetic data obtained in the reversible and irreversible binding regimes of E sigma(70) RNA polymerase (R)-lambda P-R promoter (P) open complex formation.