Fidaxomicin jams <i>Mycobacterium tuberculosis</i> RNA polymerase motions needed for initiation via RbpA contacts.

Abstract

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against <i>Mycobacterium tuberculosis</i> RNAP in vitro, but clinical use of Fdx is limited to treating <i>Clostridium difficile</i> intestinal infections due to poor absorption. To identify the structural determinants of Fdx binding to RNAP, we determined the 3.4 Å cryo-electron microscopy structure of a complete <i>M. tuberculosis</i> RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on <i>M. tuberculosis</i>. Additional structures define conformational states of <i>M. tuberculosis</i> RNAP between the free apo-holoenzyme 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 the Fdx binding pocket.