Abstract
Tagetitoxin (Tgt) inhibits multisubunit chloroplast, bacterial, and some eukaryotic RNA polymerases (RNAPs). A crystallographic structure of Tgt bound to bacterial RNAP apoenzyme shows that Tgt binds near the active site but does not explain why Tgt acts only at certain sites. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex. In this model, Tgt interacts with the β' subunit trigger loop (TL), stabilizing it in an inactive conformation. We show that (i) substitutions of the Arg residue of TL contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a "slow" enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt. Our studies highlight the role of TL as a target through which accessory proteins and antibiotics can alter the elongation complex dynamics.
Highlights
Antibiotic tagetitoxin inhibits bacterial RNA polymerases (RNAPs) and RNAP III from eukaryotes
To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex
We show that (i) substitutions of the Arg residue of trigger loop (TL) contacted by Tgt confer resistance to inhibitor; (ii) Tgt inhibits RNAP translocation, which requires TL movements; and (iii) paused complexes and a “slow” enzyme, in which the TL likely folds into an altered conformation, are resistant to Tgt
Summary
Antibiotic tagetitoxin inhibits bacterial RNA polymerases (RNAPs) and RNAP III from eukaryotes. To understand the Tgt mechanism, we constructed a structural model of Tgt bound to the transcription elongation complex In this model, Tgt interacts with the  subunit trigger loop (TL), stabilizing it in an inactive conformation. Tion complex [2] and core enzyme [3] bound to a fungal toxin ␣-amanitin suggest that the inhibitor acts through restricting the TL mobility These and other crystallographic data led to the widely accepted model of the nucleotide addition cycle, common for all multisubunit RNAPs, wherein the metamorphic TL undergoes a sequence of structural transitions, most notably between a substrate-free partially unstructured random coil state and the NTP-bound ␣-helical hairpin state (trigger helices, THs), with every nucleotide added to the nascent RNA (1, 4 – 8).
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