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

Mycobacterium tuberculosis is a critical threat to human health due to the increased prevalence of rifampin resistance (RMPr). Fitness defects have been observed in RMPr 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 RMPrM. tuberculosis RNAPs. We found that the less prevalent RMPr 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 RMPr 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.