structure-based-simulation-sampling-transcription-factor-protein

Structure-Based Simulation and Sampling of Transcription Factor Protein Movements along DNA from Atomic-Scale Stepping to Coarse-Grained Diffusion

One-dimensional (1-D) sliding of transcription factor (TF) protein along DNA is essential for facilitated diffusion of the TF to locate target DNA site ...

Beijing Computational Science Research Center

Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucleotide addition cycle. The kinetics and energetics of the process as well as how it proceeds with substantial conformational changes of the polymerase complex determine the mechano-chemical coupling mechanism of the transcription elongation. Here we investigated detailed dynamics of the PPi release process in a single-subunit RNA polymerase (RNAP) from bacteriophage T7, implementing all-atom molecular dynamics (MD) simulations. We obtained a jump-from-cavity kinetic model of the PPi release utilizing extensive nanosecond MD simulations. We found that the PPi release in T7 RNAP is initiated by the PPi dissociation from two catalytic aspartic acids, followed by a comparatively slow jump-from-cavity activation process. Combining with a number of microsecond long MD simulations, we also found that the activation process is hindered by charged residue associations as well as by local steric and hydrogen bond interactions. On the other hand, the activation is greatly assisted by a highly flexible lysine residue Lys472 that swings its side chain to pull PPi out. The mechanism can apply in general to single subunit RNA and DNA polymerases with similar molecular structures and conserved key residues. Remarkably, the flexible lysine or arginine residue appears to be a universal module that assists the PPi release even in multi-subunit RNAPs with charge facilitated hopping mechanisms. We also noticed that the PPi release is not tightly coupled to opening motions of an O-helix on the fingers domain of T7 RNAP according to the microsecond MD simulations. Our study thus supports the Brownian ratchet scenario of the mechano-chemical coupling in the transcription elongation of the single-subunit polymerase. 

A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation.

Nucleotide selection is crucial for transcription fidelity control, in particular, for viral T7 RNA polymerase (RNAP) lack of proofreading activity. It has been recognized that multiple kinetic checkpoints exist prior to full nucleotide incorporation. In this work, we implemented intensive atomistic molecular dynamics (MD) simulations to quantify how strong the nucleotide selection is at the initial checkpoint of an elongation cycle of T7 RNAP. The incoming nucleotides bind into a preinsertion site where a critical tyrosine residue locates nearby to assist the nucleotide selection. We calculated the relative binding free energy between a noncognate nucleotide and a cognate one at a preinsertion configuration via alchemical simulations, showing that a small selection free energy or the binding free energy difference (∼3 kT) exists between the two nucleotides. Indeed, another preinsertion configuration favored by the noncognate nucleotides was identified, which appears to be off path for further nucleotide insertion and additionally assists the nucleotide selection. By chemical master equation (CME) approach, we show that the small selection free energy at the preinsertion site along with the off-path noncognate nucleotide filtering can help substantially to reduce the error rate and to maintain the elongation rate high in the T7 RNAP transcription.

Nucleotide Selectivity at a Preinsertion Checkpoint of T7 RNA Polymerase Transcription Elongation.

Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA during transcription elongation by implementing extensive all-atom molecular dynamics (MD) simulations to construct a Markov state model (MSM). Our studies show that translocation proceeds in a Brownian motion, and the RNAP thermally transits among multiple metastable states. We observed non-synchronized backbone movements of the nucleic acid (NA) chains with the RNA translocation accomplished first, while the template DNA lagged. Notably, both the O-helix and Y-helix on the fingers domain play key roles in facilitating NA translocation through the helix opening. The helix opening allows a key residue Tyr639 to become inserted into the active site, which pushes the RNA-DNA hybrid forward. Another key residue, Phe644, coordinates the downstream template DNA motions by stacking and un-stacking with a transition nucleotide (TN) and its adjacent nucleotide. Moreover, the O-helix opening at pre-translocation (pre-trans) likely resists backtracking. To test this hypothesis, we computationally designed mutants of T7 RNAP by replacing the amino acids on the O-helix with counterpart residues from a mitochondrial RNAP that is capable of backtracking. The current experimental results support the hypothesis.

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

3'3'-cyclic GMP-AMP (3'3'-cGAMP) belongs to a family of the bacterial secondary messenger cyclic dinucleotides. It was first discovered in the Vibrio cholerae seventh pandemic strains and is involved in efficient intestinal colonization and chemotaxis regulation. Phosphodiesterases (PDEs) that degrade 3'3'-cGAMP play important regulatory roles in the relevant signaling pathways, and a previous study has identified three PDEs in V. cholerae, namely, V-cGAP1, V-cGAP2, and V-cGAP3, functioning in 3'3'-cGAMP degradation. We report the crystal structure, biochemical, and structural analyses of V-cGAP3, providing a foundation for understanding the mechanism of 3'3'-cGAMP degradation and regulation in general. Our crystal and molecular dynamic (MD)-simulated structures revealed that V-cGAP3 contains tandem HD-GYP domains within its N- and C-terminal domains, with similar three-dimensional topologies despite their low-sequence identity. Biochemical and structural analyses showed that the N-terminal domain plays a mechanism of positive regulation for the catalytic C-terminal domain. We also demonstrated that the other homologous Vibrio PDEs, V-cGAP1/2, likely function via a similar mechanism.

Novel Mechanism for Cyclic Dinucleotide Degradation Revealed by Structural Studies of Vibrio Phosphodiesterase V-cGAP3.

An elongation cycle of a transcribing RNA polymerase (RNAP) usually consists of multiple kinetics steps, so there exist multiple kinetic checkpoints where non-cognate nucleotides can be selected against. We conducted comprehensive free energy calculations on various nucleotide insertions for viral T7 RNAP employing all-atom molecular dynamics simulations. By comparing insertion free energy profiles between the non-cognate nucleotide species (rGTP and dATP) and a cognate one (rATP), we obtained selection free energetics from the nucleotide pre-insertion to the insertion checkpoints, and further inferred the selection energetics down to the catalytic stage. We find that the insertion of base mismatch rGTP proceeds mainly through an off-path along which both pre-insertion screening and insertion inhibition play significant roles. In comparison, the selection against dATP is found to go through an off-path pre-insertion screening along with an on-path insertion inhibition. Interestingly, we notice that two magnesium ions switch roles of leave and stay during the dATP on-path insertion. Finally, we infer that substantial selection energetic is still required to catalytically inhibit the mismatched rGTP to achieve an elongation error rate ∼10-4 or lower; while no catalytic selection seems to be further needed against dATP to obtain an error rate ∼10-2.

Determining selection free energetics from nucleotide pre-insertion to insertion in viral T7 RNA polymerase transcription fidelity control.

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.

A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control.

Cas1 and Cas2 are highly conserved proteins across clustered-regularly-interspaced-short-palindromic-repeat-Cas systems and play a significant role in protospacer acquisition. Based on crystal structure of twofold symmetric Cas1-Cas2 in complex with dual-forked protospacer DNA (psDNA), we conducted all-atom molecular dynamics simulations to study the psDNA binding, recognition, and response to cleavage on the protospacer-adjacent-motif complementary sequence, or PAMc, of Cas1-Cas2. In the simulation, we noticed that two active sites of Cas1 and Cas1' bind asymmetrically to two identical PAMc on the psDNA captured from the crystal structure. For the modified psDNA containing only one PAMc, as that to be recognized by Cas1-Cas2 in general, our simulations show that the non-PAMc association site of Cas1-Cas2 remains destabilized until after the stably bound PAMc being cleaved at the corresponding association site. Thus, long-range correlation appears to exist upon the PAMc cleavage between the two active sites (∼10 nm apart) on Cas1-Cas2, which can be allosterically mediated by psDNA and Cas2 and Cas2' in bridging. To substantiate such findings, we conducted repeated runs and further simulated Cas1-Cas2 in complex with synthesized psDNA sequences psL and psH, which have been measured with low and high frequency in acquisition, respectively. Notably, such intersite correlation becomes even more pronounced for the Cas1-Cas2 in complex with psH but remains low for the Cas1-Cas2 in complex with psL. Hence, our studies demonstrate that PAMc recognition and cleavage at one active site of Cas1-Cas2 may allosterically regulate non-PAMc association or even cleavage at the other site, and such regulation can be mediated by noncatalytic Cas2 and DNA protospacer to possibly support the ensued psDNA acquisition.

Allosteric regulation in CRISPR/Cas1-Cas2 protospacer acquisition mediated by DNA and Cas2.

In this work, we computationally investigated how a viral RNA polymerase (RNAP) from bacteriophage T7 evolves into RNAP variants under lab-directed evolution to switch recognition from T7 promoter to T3 promoter in transcription initiation. We first constructed a closed initiation complex for the wild-type T7 RNAP and then for six mutant RNAPs discovered from phage-assisted continuous evolution experiments. All-atom molecular dynamics simulations up to 1 μs each were conducted on these RNAPs in a complex with the T7 and T3 promoters. Our simulations show notably that protein-DNA electrostatic interactions or stabilities at the RNAP-DNA promoter interface well dictate the promoter recognition preference of the RNAP and variants. Key residues and structural elements that contribute significantly to switching the promoter recognition were identified. Followed by a first point mutation N748D on the specificity loop to slightly disengage the RNAP from the promoter to hinder the original recognition, we found an auxiliary helix (206-225) that takes over switching the promoter recognition upon further mutations (E222K and E207K) by forming additional charge interactions with the promoter DNA and reorientating differently on the T7 and T3 promoters. Further mutations on the AT-rich loop and the specificity loop can fully switch the RNAP-promoter recognition to the T3 promoter. Overall, our studies reveal energetics and structural dynamics details along an exemplary directed evolutionary path of the phage RNAP variants for a rewired promoter recognition function. The findings demonstrate underlying physical mechanisms and are expected to assist knowledge and data learning or rational redesign of the protein enzyme structure function.

Article	Year
A Jump-from-Cavity Pyrophosphate Ion Release Assisted by a Key Lysine Residue in T7 RNA Polymerase Transcription Elongation. PLoS computational biology\| PubMed ID: 26599007	2015
Nucleotide Selectivity at a Preinsertion Checkpoint of T7 RNA Polymerase Transcription Elongation. The journal of physical chemistry. B\| PubMed ID: 28199109	2017
T7 RNA polymerase translocation is facilitated by a helix opening on the fingers domain that may also prevent backtracking. Nucleic acids research\| PubMed ID: 28575393	2017
Novel Mechanism for Cyclic Dinucleotide Degradation Revealed by Structural Studies of Vibrio Phosphodiesterase V-cGAP3. Journal of molecular biology\| PubMed ID: 30365951	2018
Determining selection free energetics from nucleotide pre-insertion to insertion in viral T7 RNA polymerase transcription fidelity control. Nucleic acids research\| PubMed ID: 30916310	2019
A Viral T7 RNA Polymerase Ratcheting Along DNA With Fidelity Control. Computational and structural biotechnology journal\| PubMed ID: 31193497	2019
Allosteric regulation in CRISPR/Cas1-Cas2 protospacer acquisition mediated by DNA and Cas2. Biophysical journal\| PubMed ID: 34197800	2021
Switching promotor recognition of phage RNA polymerase in silico along lab-directed evolution path. Biophysical journal\| PubMed ID: 35031277	2022

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