Fidaxomicin jams M. tuberculosis RNA polymerase motions needed for initiation via RbpA contacts

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective againstMycobacterium tuberculosisRNAPin vitro, but clinical use of Fdx is limited to treatingClostridium difficileintestinal infections due to poor absorption. To enable structure-guided optimization of Fdx to treat tuberculosis, we report the 3.4 Å cryo-electron microscopy structure of a completeM. tuberculosisRNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin and explains its strong effect onM. tuberculosis. We present additional structures that define conformational states ofM. tuberculosisRNAP between the free apo-holenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on Fdx.