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

Abstract

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile 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 M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on M. tuberculosis. Additional structures define conformational states of M. tuberculosis 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.

Eliminating effects of particle adsorption to the air/water interface in single-particle cryo-electron microscopy: Bacterial RNA polymerase and CHAPSO

“…In the single particle analyses under conditions where the RNAP complexes are adsorbed to air/water interfaces and oriented, we sometimes observe an orientation A number of cryo-EM structures of bacterial RNAP transcription complexes have benefitted from CHAPSO [7,[12][13][14][15]. The observation of specifically-bound CHAPSO molecules on the Eco RNAP surface (Fig.…”

“…1, 3). These properties greatly facilitate high-resolution structure determination of these complexes using modern cryo-EM approaches [7,[12][13][14][15]].…”

The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis

“…However, the structural details between 1.1 domains from different organisms vary widely. Currently, three types of known structures of this domain exist: (i) the highly similar structures, as solved for B. subtilis and E. coli, consisting of three helices (HI to HIII) packed in antiparallel manner (8,10); (ii) the structure from T. maritima, where HI packs perpendicularly to HII and HIII (9); and (iii) the possibly disordered and flexible structure from mycobacteria with only the A N-helix discernible (11)(12)(13)(14).…”

Computational Saturation Mutagenesis to predict structural consequences of systematic mutations on protein stability and rifampin interactions in the β subunit of RNA polymerase inMycobacterium leprae

“…Given the absence of a rapid and experimentally validated system to read the impact of mutations in the -subunit of RNAP in M. leprae with clinical rifampin resistance outcomes in leprosy, we conducted computational saturation mutagenesis to determine regions on the  subunit that impact the overall stability, protein-subunit interfaces, protein-nucleic and proteinligand affinities. Being a part of the complex transcriptional machinery in the mycobacterial cell, the compositional and conformational stability of the -subunit is crucial to binding of DNA template and synthesis of complementary RNA transcript in the active center cleft of the holoenzyme (Lin et al, 2017;Boyaci et al, 2018). As rifampin blocks the growing RNA transcript through steric occlusion, its binding and orientation in the binding pocket are vital to its function (Lin et al, 2017).…”