Trigger loop of RNA polymerase is a positional, not acid–base, catalyst for both transcription and proofreading

Significance Synthesis of new RNA and removal of incorrect nucleotides during proofreading by RNA polymerase involve the transfer of two protons. Here, we show that a polymerase component, the trigger loop, does not directly mediate proton transfer during these reactions, as previously proposed. Instead, the trigger loop plays a central role in transcription as a positional catalyst, by orienting the reactants and promoting the polymerase backtracking necessary for proofreading. This positional-catalyst model of trigger-loop function explains its diverse effects on polymerase catalysis and reconciles contradictory reports in the literature. By establishing that the trigger loop is not an acid–base catalyst, our results also guide the search for alternative proton donors and acceptors for reactions in the active site of polymerase.