Article

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

ABSTRACT

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.

Download full-text

Full-text

Available from: Murali Palangat
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jul 2015 · Molecular cell
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Author Summary Mistakes made during the synthesis of messenger RNAs have been difficult to detect, both because mRNAs can be short lived, and because the translation of mRNAs into proteins has a much higher error rate that masks transcription errors. We present here a highly sensitive genetic screen that detects transcription errors and use it to identify mutations that increase the error rate of RNA polymerase II. The screen incorporates a new principle that allows transient transcription errors to cause permanent genetic changes. The screen is based on suppression of a missense mutation (cre-Y324C) in the active site of the Cre recombinase. Infrequent and transient transcription errors that restore the original codon for Y324 cause the Cre-dependent activation of a reporter gene. Background from translation errors is negligible because Cre acts as a tetramer in which all four subunits require the active site tyrosine. Transcription errors as low as ∼10−6 can be detected. We identify rpb1 mutations that define four classes, those that have increased (1) misincorporation, (2) extension of a misincorporated base, (3) both misincorporation and extension, and (4) those that block the activity of the transcription proofreading factor, TFIIS.
    Full-text · Article · Sep 2014 · PLoS Genetics
  • Source
    • "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. "
    Article: On the move
    [Show abstract] [Hide abstract]
    ABSTRACT: Single-molecule experiments have shed new light on the mechanisms responsible for the movement of RNA polymerase along DNA during transcription.
    Full-text · Article · Sep 2013 · eLife Sciences
Show more