Source of the Fitness Defect in Rifamycin-Resistant Mycobacterium tuberculosis RNA Polymerase and the Mechanism of Compensation by Mutations in the β′ Subunit

ABSTRACT Mycobacterium tuberculosis is a critical threat to human health due to the increased prevalence of rifampin resistance (RMP r ). Fitness defects have been observed in RMP r mutants with amino acid substitutions in the β subunit of RNA polymerase (RNAP). In clinical isolates, this fitness defect can be ameliorated by the presence of secondary mutations in the double-psi β-barrel (DPBB) domain of the β′ subunit of RNAP. To identify factors contributing to the fitness defects observed in vivo , several in vitro RNA transcription assays were utilized to probe initiation, elongation, termination, and 3′-RNA hydrolysis with the wild-type and RMP r M. tuberculosis RNAPs. We found that the less prevalent RMP r mutants exhibit significantly poorer termination efficiencies relative to the wild type, an important factor for proper gene expression. We also found that several mechanistic aspects of transcription of the RMP r mutant RNAPs are impacted relative to the wild type. For the clinically most prevalent mutant, the βS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the β′ subunit.