Dissociation Kinetics of Complexes between the Antibiotic Rifamycin and DNA‐Dependent RNA Polymerase from Escherichia coli

Abstract

Phosphocellulose chromatography was employed to measure the binding of 3-(2-[14C]acetamidoethyl)-thiorifamycin ([14C]AcNHEtS-Rif) to RNA polymerase core enzyme. A correct value for the stoichiometry of interaction was obtained. The same method was applied to the study of the dissociation kinetics of the [14C]AcNHEtS-Rif.cntdot.core-polymerase complex. Biphasic dissociation kinetics not in agreement with the existence of 1 single 1st order dissociation step were observed. Assuming 2 independent dissociation reactions, Kd were evaluated differing roughly by 10-fold (1.7 .times. 10-3 s-1 and 1.5 .times. 10-4 s-1 at 25.degree. C). The ratio of the amplitudes of the biphasic dissociation kinetics changed with temperature. The kinetic data suggested the existence of 2 enzyme species differing in their dynamic properties for the binding of rifamycin and indicated that the 2 enzyme forms are in equilibrium. The observed sigmoidal temperature dependence of the dissociation rate may indicate a conformational transition of RNA core polymerase with a transition midpoint at .apprx. 20.degree. C. The dye rose bengal was as effective as AcNHEtS-Rif as a competing agent. The dissociation kinetics of the [14C]-AcNHEtS-Rif.cntdot.core-polymerase complex in the presence of excess rose bengal were very similar to those measured in the presence of AcNHEtS-Rif. Rose bengal and AcNHEtS-Rif probably compete for the same site at RNA core polymerase. The dissociation of the ternary complex [14C]AcNHEtS-Rif.cntdot.core-polymerase.cntdot.poly[d(A-T)] was followed by gel filtration. Up to the extent of dissociation measured, the reaction appeared to follow 1st-order kinetics. Kd was calculated to be 1.7 .times. 10-4 s-1. The effect of streptolydigin on the dissociation kinetics of the [14C]AcNHEtS-Rif.cntdot.core-polymerase complex was not conclusive.