Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the beta subunit of Escherichia coli RNA polymerase

Abstract

Mutations to rifampicin resistance (RifR) in Escherichia coli alter the beta subunit of RNA polymerase (RNAP). A series of new point RifR mutations was isolated with the aid of random polymerase chain reaction-mediated mutagenesis followed by selection on rifampicin (Rif). All of the new RifR mutants, including changes in two novel positions, fell into the two principal clusters previously identified in the middle section of beta. Disruption of the spacer between the two clusters with deletions and insertions led to RNAP that was functional and sensitive to Rif in vitro, indicating that the spacer is not directly involved in Rif binding. However, most of the spacer mutants were strongly resistant to streptolydigin, suggesting that this area is involved in the binding of streptolydigin. An insertion of six consecutive histidines into the spacer was constructed that could be used to anchor RNAP on a Nichelate matrix without the loss of enzymatic activity, indicating that this region is looped out of the RNAP molecule.