T7 RNA polymerase translocation is facilitated by a helix opening on the fingers domain that may also prevent backtracking

Da, Lin-Tai; Chao, E; Yao, Shuai; Wu, Sudong; Su, Xiao‐Dong; Jin, Yu

doi:10.1093/nar/gkx495

Cited by 30 publications

(37 citation statements)

References 69 publications

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“…We have recently studied transcription elongation of T7 RNAP by combining physical modeling and all-atom MD simulations, addressing both mechano-chemical coupling and fidelity control mechanisms during elongation [ [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] ]. The mechanochemistry concerns about how the protein machine utilizes chemical free energy to generate mechanical or directional motions, referring to how the chemical synthesis of RNA couples with the polymerase translocation along DNA in the RNAP system.…”

Section: T7 Rna Polymerase As a Minimal Transcription Machine Model Smentioning

confidence: 99%

“…For systems of such a size, one can routinely simulate up to several microseconds under current high-performance computing technologies; yet it is still computationally prohibiting to further approach over tens of microseconds to millisecond time scale. Fortunately, by launching extensive sub-microseconds equilibrium simulations that spread around a wide range of conformation space for the relevant process, we were able to construct the Markov-state model (MSM) for the PPi release, and later for the translocation of T7 RNAP, which are estimated to happen at tens of microsecond time scale [ 31 , 34 ]. The strength and technical issues in building the MSM using MD can be found in abundant literature elsewhere [ [46] , [47] , [48] , [49] , [50] ].…”

Section: Ppi Product Release Unlikely Drives the Translocation Of T7 mentioning

confidence: 99%

“…(C) The six-state MSM of the RNAP translocation on the DNA (129 × 80 ns all-atom MD simulation, clustering ~ 9 × 10 5 conformations into 500 microstates etc.) [ 34 ]. The translocation starts after the PPi release from the product complex, or the pre-translocation state (S1), transiting all the way (via S2-S5 and mainly S3) to the post-translocation state (S6; PDB: 1MSW ) [ 16 ] (populations and transition rates are labeled).…”

Section: Ppi Product Release Unlikely Drives the Translocation Of T7 mentioning

confidence: 99%

“…The RNA and template DNA nucleotides are colored in blue and red, respectively. (D) The probability distributions of the rotational angles of the O-helix and the Y-helix during translocation process (from S1 to S6) are presented (as taken from [ 34 ]). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)…”

Section: Ppi Product Release Unlikely Drives the Translocation Of T7 mentioning

confidence: 99%

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A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control

Long

Chao

et al. 2019

Computational and Structural Biotechnology Journal

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RNA polymerase (RNAP) from bacteriophage T7 is a representative single-subunit viral RNAP that can transcribe with high promoter activities without assistances from transcription factors. We accordingly studied this small transcription machine computationally as a model system to understand underlying mechanisms of mechano-chemical coupling and fidelity control in the RNAP transcription elongation. Here we summarize our computational work from several recent publications to demonstrate first how T7 RNAP translocates via Brownian alike motions along DNA right after the catalytic product release. Then we show how the backward translocation motions are prevented at post-translocation upon successful nucleotide incorporation, which is also subject to stepwise nucleotide selection and acts as a pawl for “selective ratcheting”. The structural dynamics and energetics features revealed from our atomistic molecular dynamics (MD) simulations and related analyses on the single-subunit T7 RNAP thus provided detailed and quantitative characterizations on the Brownian-ratchet working scenario of a prototypical transcription machine with sophisticated nucleotide selectivity for fidelity control. The presented mechanisms can be more or less general for structurally similar viral or mitochondrial RNAPs and some of DNA polymerases, or even for the RNAP engine of the more complicated transcription machinery in higher organisms.

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Section: T7 Rna Polymerase As a Minimal Transcription Machine Model Smentioning

confidence: 99%

Section: Ppi Product Release Unlikely Drives the Translocation Of T7 mentioning

confidence: 99%

Section: Ppi Product Release Unlikely Drives the Translocation Of T7 mentioning

confidence: 99%

Section: Ppi Product Release Unlikely Drives the Translocation Of T7 mentioning

confidence: 99%

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A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control

Long

Chao

et al. 2019

Computational and Structural Biotechnology Journal

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“…In case that comparatively long or extensive MD simulations have been conducted, one recent study concentrates on association processes of a chromatin protein with DNA (29), but not yet the protein movements. For exemplary all-atom simulation studies on the protein movements along DNA, however, the proteins of concerns are motor proteins such as RNA polymerases (30,31), or the single-stranded DNA-binding protein (32). In this work, we focus on a model TF and present all-atom microseconds equilibrium simulations of the diffusion dynamics of the TF protein along the double stranded (ds) DNA.…”

Section: Introductionmentioning

confidence: 99%

Revealing Atomic-scale Molecular Diffusion of a Plant Transcription Factor WRKY domain protein along DNA

Dai

et al. 2020

Preprint

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designed the study. Liqiang Dai conducted the MD simulations and coarse-grained simulations and analyzed the related data. Yongping Xu solved the crystal structure of WRKY-DNA complex and conducted ITC experiment. Zhenwei Du conducted the single-molecule fluorescence experiments of WRKY diffusing on DNA. AbstractTranscription factor (TF) target search on genome is highly essential for gene expression and regulation. High-resolution determination of TF diffusion along DNA remains technically challenging. Here we constructed a TF model system of the plant WRKY domain protein in complex with DNA from crystallography and demonstrated microsecond diffusion dynamics of WRKY on the DNA employing all-atom molecular dynamics (MD) simulations. Notably, we found that WRKY preferentially binds to the Crick strand of DNA with significantly stronger energetic association than to the Watson strand. The preferential binding becomes highly prominent from non-specific to specific DNA binding, but less distinct from static binding to diffusive movements of WRKY on the DNA. Remarkably, without employing acceleration forces or bias, we captured a complete one-base pair (bp) stepping cycle of WRKY tracking along major groove of DNA with homogenous (AT)n sequence, as individual protein-DNA contacts break and reform at the binding interface. Continuous tracking of WRKY forward or backward, with occasional sliding as well as strand crossing to the minor groove of DNA, have also been captured in the simulation. The processive diffusion of WRKY had been confirmed by accompanied single-molecule fluorescence assays and coarsegrained (CG) structural simulations. The study thus provides unprecedented structural dynamics details on the TF diffusion, suggests how TF possibly approaches to gene target, and supports further high-precision experimental follow-up. The stochastic movements revealed in the TF diffusion also provide general clues on how other nucleic acid walkers step and slide along DNA. Significance StatementHow transcription factors search for target genes impact on how quickly and accurately the genes are transcribed and expressed. To locate target sufficiently fast, 1D diffusion of the protein along DNA appears essential. Experimentally, it remains challenging to determine diffusional steps of protein on DNA. Here, we report all-atom equilibrium simulations of a WRKY protein binding and diffusing on DNA, revealing structural dynamics details which have not been identified previously. We unprecedently demonstrate a complete stepping cycle of the protein for one base pair on DNA within microseconds, along with stochastic stepping or sliding, directional switching, and strand crossing. Additionally, we have found preferential DNA strand association of WRKY. These suggest how protein factors approach toward target DNA sequences.

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Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

Geronimo

Vidossich

Donati

et al. 2021

WIREs Comput Mol Sci

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DNA and RNA polymerases (Pols) are central to life, health, and biotechnology because they allow the flow of genetic information in biological systems. Importantly, Pol function and (de)regulation are linked to human diseases, notably cancer (DNA Pols) and viral infections (RNA Pols) such as COVID‐19. In addition, Pols are used in various applications such as synthesis of artificial genetic polymers and DNA amplification in molecular biology, medicine, and forensic analysis. Because of all of this, the field of Pols is an intense research area, in which computational studies contribute to elucidating experimentally inaccessible atomistic details of Pol function. In detail, Pols catalyze the replication, transcription, and repair of nucleic acids through the addition, via a nucleotidyl transfer reaction, of a nucleotide to the 3′‐end of the growing nucleic acid strand. Here, we analyze how computational methods, including force‐field‐based molecular dynamics, quantum mechanics/molecular mechanics, and free energy simulations, have advanced our understanding of Pols. We examine the complex interaction of chemical and physical events during Pol catalysis, like metal‐aided enzymatic reactions for nucleotide addition and large conformational rearrangements for substrate selection and binding. We also discuss the role of computational approaches in understanding the origin of Pol fidelity—the ability of Pols to incorporate the correct nucleotide that forms a Watson–Crick base pair with the base of the template nucleic acid strand. Finally, we explore how computations can accelerate the discovery of Pol‐targeting drugs and engineering of artificial Pols for synthetic and biotechnological applications. This article is categorized under: Structure and Mechanism > Reaction Mechanisms and Catalysis Structure and Mechanism > Computational Biochemistry and Biophysics Software > Molecular Modeling

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T7 RNA polymerase translocation is facilitated by a helix opening on the fingers domain that may also prevent backtracking

Cited by 30 publications

References 69 publications

A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control

A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control

Revealing Atomic-scale Molecular Diffusion of a Plant Transcription Factor WRKY domain protein along DNA

Computational investigations of polymerase enzymes: Structure, function, inhibition, and biotechnology

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