Interaction between Rifampicin and DNA-dependent RNA Polymerase of E. coli

Abstract

INTRODUCTION
The antibiotic rifampicin specifically inhibits bacterial RNA polymerase by blocking the initiation, but not the elongation, of RNA chains (Sippel and Hartmann 1968). As shown earlier, rifampicin forms a tight 1:1 complex with the enzyme (Wehrli et al. 1968a; Wehrli and Staehelin 1970; Eilen and Krakow 1973). Therefore very low concentrations of antibiotic are required to inhibit the enzyme. Mammalian RNA polymerases, as well as other nucleic acid polymerases of both bacterial and mammalian origin, are not affected at these low levels of rifampicin (Wehrli et al. 1968b; Wehrli and Staehelin 1975). Thus rifampicin is a very specific and powerful inhibitor and has been extensively used in the study of RNA biosynthesis and metabolism. Although it has been suggested that the strength of rifampicin binding to RNA polymerase is correlated to the extent of enzyme inhibition (Wehrli and Staehelin 1971), no direct quantitative measurements of this binding have been made.

In this paper we describe a new method that allows both the rate of association and the rate of dissociation of rifampicin and RNA polymerase to be measured directly. The application of this method makes it possible to quantify the kinetic interaction of rifampicin not only with the free holo- or core enzyme under a variety of physical conditions, but also with RNA polymerase engaged in the various steps of transcription.

MATERIALS AND METHODS
[¹⁴C] Rifampicin (11.6 μCi/μmole) was prepared as previously described (Keberle, Meyer-Brunot and Schmid 1966). The preparation of dextran-coated charcoal, the isolation of the rifampicin–RNA...