RFA1 Inhibits Rifampicin-resistant RNA Polymerase by a Similar Mechanism as Rifampicin.

The anti-tuberculosis agent rifampicin functions by binding to RNA polymerase (RNAP) around the active centre Mg2+ and sterically prevents the synthesis of RNA products greater than 2-3 nt in length. In the rifampicin-resistant TB strains, the amino acid residues at/near the rifampicin-binding site on RNAP are substituted by other residues. RFA1, a rifabutin analogue, shows activity against the rifampicin-resistant TB strains. We show that RFA1 inhibits transcription initiation by RNAP, as well as by rifampicin-resistant polymerase derivatives. Like rifampicin, RFA1 does not inhibit transcription elongation once the transcript length reaches 3 nt or beyond. RFA1-resistant substitutions that are located at the β-subunit of RNAP, 40-45 Å away from the active centre Mg2+, impair the binding and function of RFA1, and to a lesser extent, affect rifampicin activity. A higher concentration of Mg2+ is detrimental to RFA1-mediated transcription inhibition. Our results establish that RFA1 is an effective inhibitor of bacterial transcription and could be a potential drug candidate for the treatment of TB and rifampicin-resistant TB once its pharmacokinetics are established.