Influence of Temperature on the Action of Rifampicin on RNA Polymerase in Presence of DNA

When rifampicin, together with ribonucleoside triphosphates, is added at 25 °C to a mixture of RNA polymerase and DNA which had been preincubated for a few minutes, RNA is synthesized despite the presence of the inhibitor. With 0.8 mM Mg2+ present during the preincubation an optimum of RNA synthesis is observed which equals the synthesis in the absence of rifampicin. The extent of RNA synthesis at 25 °C is dependent on the temperature at which the enzyme has been preincubated with DNA. RNA synthesis in the presence of the inhibitor increases almost linearly with preincubation temperatures from 5 to 25 °C. The same results are obtained by applying either the chain initiation assay or the chain elongation assay for measuring RNA synthesis. The effect of temperature during preincubation of enzyme and template on subsequent RNA synthesis at 25 °C proved to be reversible. Experiments with poly[d(A‐T)] as competing template are consistent with the notion that during preincubation RNA polymerase is bound to the template at low as well as at higher temperatures under the conditions used. Essentially the same results concerning the initiation of RNA synthesis in the presence of rifampicin are obtained with T4 DNA, T2 DNA and poly[d(A‐T)] as templates. These results support the hypothesis that RNA polymerase bound to DNA exists in at least two different states which differ in their susceptibility to rifampicin. These different states are in a rather rapid and temperature‐dependent equilibrium. According to this hypothesis rifampicin prevents the conversion of the sensitive state of the complex of RNA polymerase and DNA to the resistant form. Subsequent initiation of RNA synthesis by addition of substrate is consequently blocked.