The overall goal of this study is to dissect the catalytic center and the regulatory domain(s) of RNA polymerase of Escherichia coli to study the mechanisms of transcription. In particular, the effect on different steps of transcription of mutations in RNA polymerase that confer rifampicin resistance (rif) have been analyzed. The antibiotic rifampicin inhibits transcription of RNA polymerase from all eubacteria indicating that the rifampicin binding site(s) is in a region that may be conserved and important for RNA polymerase functions. RNA polymerases with altered rifampicin binding are likely to be also altered in some vital transcription processes. By studying the correlation between a particular rif RNA polymerase and its altered transcription property, one can assign the functional role of the rifampicin binding site(s) of RNA polymerase. Some of rif RNA polymerase are found to be defective in promoter clearance (a transition step in transcription at which an RNA polymerase exits from the initiation stage to the elongation stage) and to enhance abortive initiation products suggesting that the rifampicin binding site(s) is involved in this important process. The mechanisms underlying the defect in promoter clearance of one rif RNA polymerase in which there is an Arg 529 Cys change in the beta subunit have been studied in detail both in vivo and in vitro. This mutant RNA polymerase is found to have reduced affinity for a nucleoside triphosphates during initiation and is subject to a high Km barrier during promoter clearance process. Since this rif RNA polymerase also has reduced affinity for the same nucleoside triphosphates during elongation, it is plausible that the amino acid residue Arg 529 of the beta subunit is part of the catalytic center of RNA polymerase.
