The transcription inhibitor lipiarmycin blocks DNA fitting into the RNA polymerase catalytic site

Abstract

Worldwide spreading of drug-resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the δ70 subunit region 3.2 and the RNAP β′ subunit switch-2 element, which controls the clamping of promoter DNA in the RNAP active-site cleft. Lpm abolishes isomerization of the 'closed'-promoter complex to the transcriptionally competent 'open' complex and blocks δ70- stimulated RNA synthesis on promoter-less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA-strand fitting into the RNAP active-site cleft directed by the β′ subunit switch-2 element and the δ 70 subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process. © 2010 European Molecular Biology Organization | All Rights Reserved.