RNA Polymerase II Translocation and Fidelity are Governed by Trigger Loop Dynamics: A Single-Molecule Study

Abstract

RNA polymerase II (RNAPII) carries out the transcription of all mRNA in eukaryotic cells. During transcript elongation, RNAPII must select the correct nucleotide, catalyze its addition to the growing RNA chain, and move stepwise along the template DNA until the gene is fully transcribed. The trigger loop is an evolutionarily conserved protein subdomain that has been implicated in substrate selection and catalysis in multi-subunit RNA polymerases, but its role in the elongation process is not fully elucidated. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motions of individual wild-type and trigger-loop mutant RNAPII molecules. We report direct evidence for trigger loop involvement in the RNAPII translocation event. Global fits to the force-velocity relationships for RNAPII and its trigger loop mutants support a Brownian-ratchet model for elongation, where the incoming NTP is able to bind in either pre- or post-translocated states, and movement between these two states is governed by the trigger loop. Under conditions that promote misincorporation, the observed pausing kinetics suggest that the trigger loop governs fidelity in both substrate selection and mismatch recognition.