Rifampicin transcription effects

A high-throughput assay for bacterial RNA polymerase has been successfully developed and validated using a 96-well, automated format [70], The reaction mixture contained a DNA template, nucleotide substrates (NTPs), supplemented with a-33P-labeled CTP in Tris-acetate buffer (pH 6.8). The polymerase reaction was carried out at 34°C for 40 min (providing linear kinetics). The effect of dimethylsulfoxide (DMSO), the usual solvent for test compounds used in a screen, was taken into consideration. The radiolabeled RNA transcripts were allowed to bind diethyl aminoethyl (DEAE) beads, which were then separated via filtration, and radioactivity associated with the wells was quantitated to measure the RNA polymerase activity. The standard deviation of the measured activity was typically < 15% of the average. Use of this assay to screen for RNA polymerase inhibitors from chemical libraries and natural products led to the identification of DNA intercalators (known to inhibit RNA polymerase activity), rifampicin (a known inhibitors of RNA polymerase), and several derivatives of rifampicin from Actinomycetes extracts. Therefore this assay can be reliably utilized to detect novel inhibitors of bacterial RNA polymerase. 

From the biochemical point of view, the unique mode of action of some of the ansamycins, in particular of rifampicin, has aroused much interest. Rifampicin and several other ansamycins have been shown to inhibit bacterial transcription very specifically and at extremely low concentrations by interacting exclusively with DNA-dependent RNA polymerase. This unique action has spurred many investigations on the effects of ansamycins in a variety of viral and eukaryotic systems. More recently maytansine and related compounds have been found to be very potent antimitotic agents and to have interesting antitumour activities. 

Rifampicin was first shown by Hartmann et al. 54 to have a specific inhibitory effect on RNA polymerase from E. coli. Later, other active ansamycins were found and RNA polymerases from a large variety of bacteria other than E. coli proved to be sensitive to the drug. More recently, an RNA polymerase from E. coli containing only one subunit and probably involved in the initiation of DNA replication (dna G gene product) has been shown to be resistant to rifampicin5 s This holds true also for the various mammalian RNA polymerases. In contrast to non-specific inhibitors of transcription such as actinomycin and mitomycin, rifampicin interacts specifically with the bacterial enzyme itself. With the aid of 14C-labelled rifampicin it could be shown that the drug forms a very stable complex with the enzyme in a molar ratio of 1 1S6> 57 The dissociation constant of this complex is 10-9 M at 37 °C and... 

Rifampicin and other members of the rifamycin group bind to the subunit of bacterial RNA polymerase and block the formation of the first phosphodiester bond. They therefore inhibit initiation, with little effect on elongation or termination of previously initiated chains. Actinomycin D binds tightly to double-stranded DNA and prevents it from acting as a template for transcription. It binds between adjacent base pairs, particularly in G-rich regions, by a process known as intercalation. At low concentration, RNA synthesis is effectively inhibited with little effect on DNA or protein synthesis. 

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