Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

Biophysics Program, Stanford University, Stanford, CA 94305, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2012; 109(17):6555-60. DOI: 10.1073/pnas.1200939109
Source: PubMed


During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre- or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate.

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    • "It has been proposed by a number of groups that TL movement contributes to or controls translocation (Bar-Nahum et al., 2005; Brueckner and Cramer, 2008; Feig and Burton, 2010; Kaplan et al., 2012; Larson et al., 2012). Our structures show Pol II in a post-translocated state, with a TL in the off state due to interactions with neighboring Rpb1 residues (Figures 6A, 6B, and S6B). "
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    • "The first intrinsic mechanism includes regulation of the trigger loop movement. Interaction of Rpb1 E1103 residue with the H2 hinge (residues 1095–1099) of the trigger loop has been previously proposed to delay the trigger loop closure thus slowing down transcription elongation and supporting fidelity maintenance [3,19,23,62]. The observation that rpb1-T1113P substitution renders transcription error-prone indicates that the T1113 residue plays the same or similar role as E1103. "
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    • "Transcription | On the move Forties et al. eLife 2013;2:e01414. DOI: 10.7554/eLife.01414 2 of 3 Insight and colleagues to propose the existence of a secondary site for binding NTP (Abbondanzieri et al., 2005; Larson et al., 2012). There are good reasons to believe that NTP binding is in equilibrium: in other words, the rates at which NTP molecules bind to, and unbind from, the RNA polymerase are much faster than the catalysis rate. "
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