Thiolutin is a direct inhibitor of RNA Polymerase II

Thiolutin is a well-known and longused natural product transcription inhibitor with an unresolved mode of action. Recent studies have identified Zn2+-chelation activity for both thiolutin and the related dithiolopyrrolone holomycin, with direct inhibition of RNA polymerases ruled out as a mode of action. However, negative results for direct transcription inhibition of RNA polymerases in recent studies are contradicted by previously observed thiolutin inhibition of extract fractions containing yeast RNA Polymerases I, II, and III. Here, we present chemical genetic and biochemical approaches to investigate the mode of action of thiolutin. We identify diverse classes of mutants that alter sensitivity to thiolutin. We functionally dissect the multidrug resistance and thioredoxin pathways controlling thiolutin sensitivity. We provide genetic evidence that thiolutin causes oxidation of thioredoxins in vivo, and suggest that thiolutin induces oxidative stress and alters Zn2+, Cu2+ and Mn2+ homeostasis in vivo, recapitulating thiolutin interactions with Zn2+. Finally, our results resolve contradictory biochemical results for thiolutin inhibition of transcription by direct demonstration of thiolutin inhibition of RNA polymerase II (Pol II) in vitro. Inhibitory activity requires both appropriate reduction of thiolutin and the presence of Mn2+. Thio/Mn2+ inhibition is abrogated when template DNA is pre-bound to Pol II or when excess DTT is present, and renders Pol II pause prone in elongation if initiation is bypassed using a pre-formed Pol II elongation complex. Together, we propose that thiolutin directly inhibits Pol II transcription through a novel mechanism distinct from known transcription inhibitors.