The role of the beta-subunit of RNA-polymerase in specific recognition of promoters

The complex formation of T7 DNA with RNA polymerase from E. coli B/r WU-36-10-11-12 (E. coli W12) and its rifampicin resistant mutant with highly pleiotropic effect--rpoB409 was studied. As shown earlier rpoB409 RNA polymerase differs from the normal enzyme by the selection of RNA synthesis from early promoters of DNA from T7 and T4 phages. The change in the RNA specificity synthesis due to rpoB409 mutation was shown to occur at the stage of RNA polymerase interaction with DNA before open promoter complex formation. The data obtained together with the fact of highly pleiotropic effect of the rpoB409 mutation indicate that RNA polymerase beta-subunit takes part in specific recognition of promoters.     

Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance

Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase can confer resistance to rifampicin. A large number of rifampicin-resistant (hereafter called Rifr) mutants have been isolated in Escherichia coli to probe the involvement of RNA polymerase in a variety of physiological processes. We have undertaken a comprehensive analysis of Rifr mutations to identify their structural and functional effects on RNA polymerase. Forty-two Rifr isolates with a variety of phenotypes were mapped to defined intervals within the rpoB gene using a set of deletions of the rpoB gene. The mutations were sequenced. Seventeen mutational alterations affecting 14 amino acid residues were identified. These alleles are located in three distinct clusters in the center of the rpoB gene. We discuss the implications of our results with regards to the structure of the rifampicin binding site.     

RNA polymerase of a rifampicin-resistant mutant of Escherichia coli has an altered selectivity to phage T7 DNA promoters

The formation of complexes of RNA polymerases from E. coli W12 and its rpoB409 rifampicin resistant mutant with A1 and D promoters of T7 delta D111 DNA was studied by an abortive RNA synthesis technique. The mutation was shown to affect RNA synthesis initiation at these two promotors differentially so that the efficiency of D promotor utilization is enhanced but the use of A1 promotor is unchanged. The mutation does not interfere with the affinity of the enzyme for both initiating substrates. The results show that the change in RNA polymerase beta-subunit structure has a differential effect on the enzyme interaction with different promotors. The necessity of a classificatory approach to structure-functional analysis of promotors was proposed.