SOURCE OF THE FITNESS DEFECT IN RIFAMYCIN-RESISTANT M. TUBERCULOSIS RNA POLYMERASE AND THE MECHANISM OF COMPENSATION BY MUTATIONS IN THE {beta}'-SUBUNIT [PublishAheadOfPrint]

Mycobacterium tuberculosis (MTB) 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 having 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 MTB RNAPs. We found that the less prevalent RMP^R mutants exhibit significantly poorer termination efficiencies relative to 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 wild-type. For the clinically most prevalent, βS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the β'-subunit.

https://ift.tt/2JN0buB