RIFAMPICIN REGION REVISITED - NEW RIFAMPICIN-RESISTANT AND STREPTOLYDIGIN-RESISTANT MUTANTS IN THE BETA-SUBUNIT OF ESCHERICHIA-COLI RNA-POLYMERASE

Mutations to rifampicin resistance (Rif(R)) in Escherichia coli alter the beta subunit of RNA polymerase (RNAP). A series of new point Rif(R ) mutations was isolated with the aid of random polymerase chain react ion-mediated mutagenesis followed by selection on rifampicin (Rif). Al l of the new Rif(R) mutants, including changes in two novel positions, fell into the two principal clusters previously identified in the mid dle section of beta. Disruption of the spacer between the two clusters with deletions and insertions led to RNAP that was functional and sen sitive to Rif in vitro, indicating that the spacer is not directly inv olved in Rif binding. However, most of the spacer mutants were strongl y resistant to streptolydigin, suggesting that this area is involved i n the binding of streptolydigin. An insertion of six consecutive histi dines into the spacer was constructed that could be used to anchor RNA P on a Ni-chelate matrix without the loss of enzymatic activity, indic ating that this region is looped out of the RNAP molecule.