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

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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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    ABSTRACT: Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF-stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop 1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the trigger loop (TL), allowing visualization of its open state. Overall, our observations suggest that "open/closed" conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular cell 07/2015; 59(2):258-269. DOI:10.1016/j.molcel.2015.06.034 · 14.02 Impact Factor
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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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    • "This mechanism received further support from single-molecule studies, which followed the dynamics of individual transcription elongation complexes (TECs) (Abbondanzieri et al., 2005; Bai et al., 2007; Larson et al., 2012). Nonetheless, in order to explain the relationship between the elongation velocity and the external force applied to RNAP obtained from single-molecule experiments, the classical linear ratchet mechanism (Figure 1) had to be modified such that the incoming NTP must also bind to the pre-translocated TEC (Figure 1—figure supplement 1) (Abbondanzieri et al., 2005; Larson et al., 2012). In the pre-translocated TEC, the primary nucleotide binding site is occupied by the 3′-end of the nascent transcript. "
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