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.
Highlights
The bacterial RNA polymerase (RNAP) is a proven target for antibiotics
We compared Fdx inhibition of mycobacterial RNAPs containing core RNAP combined with A ( A-holo) and RbpA with inhibition of Escherichia coli (Eco) 70-holo ss s using a quantitative abortive initiation assay (Davis et al, 2015)
Numerous subsequent crystal structures have supported the idea that stable, transcriptioncompetent complexes of RNAP with nucleic acids, either RPo (Bae et al, 2015; Hubin et al, 2017b; Zuo and Steitz, 2015) or elongation complexes (Gnatt et al, 2001; Kettenberger et al, 2004; Vassylyev et al, 2007), correlate with the closed-clamp conformation
Summary
The bacterial RNA polymerase (RNAP) is a proven target for antibiotics. The rifamycin (Rif) class of antibiotics, which inhibit RNAP function, is a lynchpin of modern tuberculosis (TB) treatment (Chakraborty and Rhee, 2015). TB, caused by the infectious agent Mycobacterium tuberculosis (Mtb), is responsible for almost 2 million deaths a year. It is estimated that one third of the world is infected. Mortality from TB is increasing, partly due to the emergence of strains resistant to Rifs (RifR) (Zumla et al, 2015). Additional antibiotics against RifR Mtb are needed
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