RNA polymerase II flexibility during translocation from normal mode analysis

Feig, Michael; Burton, Zachary F.

doi:10.1002/prot.22560

Cited by 23 publications

(34 citation statements)

References 63 publications

(67 reference statements)

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“…Taken together, these results suggest that the TL serves to affect not only the rate of catalysis, as previously found, but also the NTP-binding affinity and the translocation equilibrium. An inhibitory effect of TL closure on translocation was recently proposed, based on structural modeling and computational studies (30), and is consistent with the proposed "two-ratchet model" for RNAP translocation (27). In that proposal, the closed state of the TL, which is dependent Table 1.…”

Section: Resultssupporting

confidence: 76%

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

Larson

Zhou

Kaplan

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

128

187

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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.optical trap | optical tweezers | Pol II T he transcription of genetic information stably encoded in DNA into a transient RNA message occurs with remarkable fidelity. RNA polymerase (RNAP) incorporates nucleotides into nascent RNA chains at rates of 10-70 s −1 (1, 2), but only inserts the wrong nucleotide approximately once per 100,000 bases, on average (3). Although the energetics of base-pairing is fundamental to transcription fidelity, the discrimination for correctly matched nucleotide substrates is kinetically controlled, and accomplished by active site conformational changes centered on the trigger loop (TL) (4-7). The TL is an evolutionarily conserved mobile element that stabilizes substrate NTPs in the active site of a variety of multisubunit RNA polymerases, including eukaryotic RNAP I, II, and III, as well as bacterial and archaeal RNAP (8). In the case of RNAPII, alteration of the TL has important consequences for activity and fidelity. TL residue His1085, for example, interacts with the bound NTP substrate, and is fully conserved among multisubunit RNA polymerases. The importance of this residue is underscored by the lethality of the H1085A substitution in yeast (7) and catalytic defects resulting from substitutions at this position for both the yeast and bacterial enzymes (7, 9-11). Additionally, a substitution for residue Glu1103 of the TL has been found not only to promote nucleotide misincorporation (6, 7), but also to increase the elongation rate (6,7,12), properties that are jointly consistent with the notion of kinetic proofreading (13). To explore further the interplay between elongation rate and transcriptional fidelity, we determined the effec...

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Section: Resultssupporting

confidence: 76%

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

Larson

Zhou

Kaplan

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

128

187

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“…Although not yet done, simulation of R428A in a structure with a relaxed trigger loop conformation (PDB 205I or 2PPB) might be much less informative, because the R428A substitution may not as strongly disrupt an already relaxed TEC. For instance, opening the trigger loop is expected to remove constraints on bridge α-helix dynamics by reducing contacts between the bridge helix and the trigger helices [9]. Based on these results and ideas, we suggest that wt simulations may require a reference structure (in this case R428A) to be most useful and that the choice of starting wt structure (in this case a strained catalytic structure) may be crucial.…”

Section: Methodsmentioning

confidence: 99%

“…In the catalytic structure, the closed trigger helices pack closely with the bridge α-helix, which is a prominent and dynamic feature of RNAP that borders the active site [1, 5, 6]. It has been suggested that bridge helix dynamics, which is expected to be regulated by trigger loop opening and closing, may provide the thermal driving force for translocation of nucleic acids through RNAP [7–9]. …”

Section: Introductionmentioning

confidence: 99%

Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

Seibold¹,

Singh²,

Zhang³

et al. 2010

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β’ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.

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“…In the two ratchet model, one ratchet is the binding of NTP substrate in the Pol II active site where catalysis of phosphodiester bond formation gives directionality to translocation, and the second ratchet relates to conformational states of the enzyme that permit or disfavor translocation (a possibility discussed in molecular dynamics studies on Pol II from Feig and Burton and coworkers [67, 68]). Occupancy of the Pol II active site by a basepaired NTP indeed stabilizes Pol II in the post-translocated state when it cannot be hydrolyzed [37].…”

Section: Pol II Dynamics and Translocationmentioning

confidence: 99%

“…A number of molecular dynamics studies have been performed on Pol II and other msRNAPs to gain insight into possible conformational changes underlying translocation [45, 59, 67, 68]. Reference [59], cited above for studies on a the BH, coupled molecular dynamics on Thermus thermophilus RNAP with extensive mutagenesis in the S. cerevisiae Pol II system and described a number of interactions that develop stably in molecular dynamics simulations and are mutually exclusively to either open or closed TL structures.…”

Section: Pol II Dynamics and Translocationmentioning

confidence: 99%

Basic mechanisms of RNA polymerase II activity and alteration of gene expression in Saccharomyces cerevisiae

Kaplan

2013

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Transcription by RNA Polymerase II (Pol II), and all RNA polymerases for that matter, may be understood as comprising two cycles. The first cycle relates to the basic mechanism of the transcription process wherein Pol II must select the appropriate nucleoside triphosphate (NTP) substrate complementary to the DNA template, catalyze phosphodiester bond formation, and translocate to the next position on the DNA template. Performing this cycle in an iterative fashion allows the synthesis of RNA chains that can be over one million nucleotides in length in some larger eukaryotes. Overlaid upon this enzymatic cycle, transcription may be divided into another cycle of three phases: initiation, elongation, and termination. Each of these phases has a large number of associated transcription factors that function to promote or regulate the gene expression process. Complicating matters, each phase of the latter transcription cycle are coincident with cotranscriptional RNA processing events. Additionally, transcription takes place within a highly dynamic and regulated chromatin environment. This chromatin environment is radically impacted by active transcription and associated chromatin modifications and remodeling, while also functioning as a major platform for Pol II regulation. This review will focus on our basic knowledge of the Pol II transcription mechanism, and how altered Pol II activity impacts gene expression in vivo in the model eukaryote Saccharomyces cerevisiae.

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RNA polymerase II flexibility during translocation from normal mode analysis

Cited by 23 publications

References 63 publications

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

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

Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

Basic mechanisms of RNA polymerase II activity and alteration of gene expression in Saccharomyces cerevisiae

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