Effects of rifampicin on synthesis and functional activity of DNA-dependent RNA polymerase in Escherichia coli

Abstract

During the course of kinetic studies on the synthesis of RNA polymerase subunits in Escherichia coli K12, strain Km7 (CP372), certain anomalies were found that seemed to be associated with the system of reversible inhibition of RNA and protein synthesis by rifampicin. To find a possible explanation for these anomalies, effects of rifampicin on RNA chain elongation and on residual synthesis of polymerase subunits were investigated with several strains including Km7. Examination of mRNA synthesis for the tryptophan operon suggested that RNA chain growth as well as RNA chain initiation is inhibited at high drug concentration (500 mug/ml), wheras RNA chain initiation is inhibited specifically at low concentration (20 mug/ml). Analysis of effect of rifampicin concentration on total RNA synthesis gave results that are also consistent with this conclusion. These results emphasize the need for selecting a proper drug concentration whenever rifampicin or other related antibiotic is used as a specific inhibitor of transcription initiation. When rifampicin was added to a culture of these strains absolute rates of synthesis of all subunits of RNA polymerase increased for several minutes and then decreased. The extent of this transient stimulation varied depending on the strain, drug concentration and other conditions, but was most striking for the beta and sigma subunits with strain Km7 at high drug concentration (500 mug/ml). With a rifampicin-sensitive wild-type strain tested, the maximum stimulation was found at about 50 mug/ml of the drug, with a particularly marked effect for sigma subunit. Streptolydigin, on the other hand, inhibited the synthesis of core subunits much faster than the bulk of protein, but inhibited synthesis of sigma subunit only after a lag. Hence a specific effect of rifampicin but not the inactivation of beta subunit per se appears to be involved in transient stimulation of polymerase synthesis observed. Implications of these findings on the control of RNA polymerase synthesis are discussed.