Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism

Dangkulwanich, Manchuta; Ishibashi, Toyotaka; Liu, Shixin; Kireeva, Maria L.; Lubkowska, Lucyna; Kashlev, Mikhail; Bustamante, Carlos

doi:10.7554/elife.00971

Cited by 124 publications

(207 citation statements)

References 73 publications

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“…The existence of the elemental pause is best documented in bacteria; it has been argued that eukaryotic RNAPII passes directly into a backtrack pause state without an elemental pause intermediate [49, 50], but current data on this point are inconclusive [41]. Compared to the bacterial enzyme, what happens in the catalytic center of RNAPII during pausing is less well understood, and hairpin-stabilization of RNAPII pausing has not been described to date.…”

Section: Nascent Rna Modulates the Activity Of Rnapmentioning

confidence: 99%

A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure

Zhang

Landick

2016

Trends in Biochemical Sciences

121

124

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The vectorial (5′-to-3′ at varying velocity) synthesis of RNA by cellular RNA polymerases creates a rugged kinetic landscape, demarcated by frequent, sometimes long-lived pauses. In addition to myriad gene-regulatory roles, these pauses temporally and spatially program the co-transcriptional, hierarchical folding of biologically active RNAs. Conversely, these RNA structures, which form inside or near the RNA exit channel, interact with the polymerase and adjacent protein factors to influence RNA synthesis by modulating pausing, termination, antitermination, and slippage. Here we review the evolutionary origin, mechanistic underpinnings, and regulatory consequences of this interplay between RNA polymerase and nascent RNA structure. We categorize and attempt to rationalize the extensive linkage between the transcriptional machinery and its product, and provide a framework for future studies.

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Section: Nascent Rna Modulates the Activity Of Rnapmentioning

confidence: 99%

A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure

Zhang

Landick

2016

Trends in Biochemical Sciences

121

124

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“…After fitting to the single-molecule experiments 61 , our KNM provides explanations to two slow steps during Pol II elongation, as identified by the single-molecule experiment 61 (Figure 5a). …”

Section: Applicationsmentioning

confidence: 99%

Elucidation of the Dynamics of Transcription Elongation by RNA Polymerase II using Kinetic Network Models

et al. 2016

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CONSPECTUS RNA polymerase II (Pol II) is an essential enzyme that catalyzes transcription with high efficiency and fidelity in eukaryotic cells. During transcription elongation, Pol II catalyzes the nucleotide addition cycle (NAC) to synthesize messenger RNA using DNA as the template. The transitions between the states of the NAC require conformational changes of both the protein and nucleotides. Although X-ray structures are available for most of these states, the dynamics of the transitions between states are largely unknown. Molecular dynamics (MD) simulations can predict structure-based molecular details and shed light on the mechanisms of these dynamic transitions. However, the employment of MD simulations on a macromolecule (tens to hundreds of nanoseconds) such as Pol II is challenging due to the difficulty of reaching biologically relevant timescales (tens of microseconds or even longer). To overcome this challenge, kinetic network models (KNMs) such as Markov State Models (MSMs) have become a popular approach to assess long-timescale conformational changes using many short MD simulations. We describe here our application of KNMs to characterize the molecular mechanisms of the NAC of Pol II. First, we introduce the general background of MSMs and further explain the procedures for the construction and validation of MSMs by providing some technical details. Next, we give an outline of our previous studies in which we applied MSMs to investigate the individual steps of the NAC, including translocation and pyrophosphate ion release. We make a summary of the major findings for each of these MSM applications. Furthermore, we describe in detail how to build the structural models, the procedures to generate conformations for seeding MD simulations and the parameters used to construct MSMs for each of the application we present. Finally, in order to study the overall NAC, we combine the individual steps of the NAC into a five-state KNM based on a non-branched Brownian ratchet scheme to explain the single-molecule optical tweezers experimental data. In the description of the KNM application, we explicitly write out the underlying assumptions of the five-state KNM and discuss the open questions and future studies that can help us refine the KNMs. The studies we discuss in the review complement experimental observations and provide molecular mechanisms for the transcription elongation cycle. In the long term, incorporation of sequence-dependent kinetic parameters into KNMs has a great potential for identifying error-prone sequences and predicting transcription dynamics in genome-wide transcriptomes.

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“…2B). In each nucleotide-addition cycle in transcription elongation, RNAP translocates between a “pre-translocated state,” in which the RNAP active-center “i” and “i+1” sites interact with RNA positions −2 and −1, and a “post-translocated state,” in which the RNAP i site interacts with RNA position −1 and the RNAP i+1 site is unoccupied and available for binding of an NTP (1,2,16). Translocation requires breaking the DNA base pair at position +1 and breaking the RNA-DNA base pair at position −10 (17-18).…”

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confidence: 99%

“…Thus, each position of the consensus sequence is predicted to favor the pre-translocated state over the post-translocated state (−10 through effects on duplex stability, −1 through effects on active-center binding, and +1 through both). Accordingly, each position of the consensus PE would be predicted to increase the opportunity for the TEC to enter an “elemental pause” state (a state that, according to one view, is accessed from the pre-translocated state and serves as an obligatory intermediate for pausing; 7,8,10-11,20) or a “backtracked” state (a state that, according to another view, is accessed from the pre-translocated state and serves as the primary state for pausing; 2,16). …”

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confidence: 99%

Interactions between RNA polymerase and the “core recognition element” counteract pausing

Vvedenskaya

Vahedian-Movahed

Bird

et al. 2014

Science

172

207

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Transcription elongation is interrupted by sequences that inhibit nucleotide addition and cause RNA polymerase (RNAP) to pause. Here, by use of native-elongating-transcript sequencing (NET-seq) and a variant of NET-seq that enables analysis of mutant RNAP derivatives in merodiploid cells (mNET-seq), we analyze transcriptional pausing genome-wide in vivo in Escherichia coli. We identify a consensus pause-inducing sequence element, G−10Y−1G+1 (where −1 corresponds to the position of the RNA 3′ end). We demonstrate that sequence-specific interactions between RNA polymerase core enzyme and a core recognition element (CRE) that stabilize transcription initiation complexes also occur in transcription elongation complexes and facilitate pause read-through by stabilizing RNAP in a post-translocated register. Our findings identify key sequence determinants of transcriptional pausing and establish that RNAP-CRE interactions modulate pausing.

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Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism

Cited by 124 publications

References 73 publications

A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure

A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure

Elucidation of the Dynamics of Transcription Elongation by RNA Polymerase II using Kinetic Network Models

Interactions between RNA polymerase and the “core recognition element” counteract pausing

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