CBR antimicrobials alter coupling between the bridge helix and the β subunit in RNA polymerase

Bacterial RNA polymerase (RNAP) is a validated target for antibacterial drugs. CBR703 series antimicrobials allosterically inhibit transcription by binding to a conserved a helix (beta' bridge helix, BH) that interconnects the two largest RNAP subunits. Here we show that disruption of the BH-beta subunit contacts by amino-acid substitutions invariably results in accelerated catalysis, slowed-down forward translocation and insensitivity to regulatory pauses. CBR703 partially reverses these effects in CBR-resistant RNAPs while inhibiting catalysis and promoting pausing in CBR-sensitive RNAPs. The differential response of variant RNAPs to CBR703 suggests that the inhibitor binds in a cavity walled by the BH, the beta' F-loop and the beta fork loop. Collectively, our data are consistent with a model in which the b subunit fine tunes RNAP elongation activities by altering the BH conformation, whereas CBRs deregulate transcription by increasing coupling between the BH and the beta subunit.