My research is mainly on physical modeling and simulation of biomolecular machinery, particularly those that regulate genetic process. I have recently studied a viral DNA packaging motor, DNA/RNA helicases, polymerases, etc.. I'm interested in understanding how these microscopic machines react to thermal fluctuations and environmental noises, how they achieve regular movements and high-fidelity functions, and how they emerge, evolve through and impact on evolution. I'm also interested in bringing biomolecular redesign from in silico to in vitro and vivo. Supervisors: George Oster and Carlos Bustamante
Homologous recombination plays a key role in the restart of stalled replication forks and in the ... more Homologous recombination plays a key role in the restart of stalled replication forks and in the generation of genetic diversity. During this process, two homologous DNA molecules undergo strand exchange to form a four-way DNA (Holliday) junction. In the presence of metal ions, the Holliday junction folds into the stacked-X structure that has two alternative conformers. Experiments have revealed the spontaneous transitions between these conformers, but their detailed pathways are not known. Here, we report a series of molecular dynamics simulations of the Holliday junction at physiological and elevated (400 K) temperatures. The simulations reveal new tetrahedral intermediates and suggest a schematic framework for conformer transitions. The tetrahedral intermediates bear resemblance to the junction conformation in complex with a junction-resolving enzyme, T7 endonuclease I, and indeed, one intermediate forms a stable complex with the enzyme as demonstrated in one simulation. We also describe free energy minima for various states of the Holliday junction system, which arise during conformer transitions. The results show that magnesium ions stabilize the stacked-X form and destabilize the open and tetrahedral intermediates. Overall, our study provides a detailed dynamic model of the Holliday junction undergoing a conformer transition.
ABSTRACTCas1 and Cas2 are highly conserved proteins across CRISPR-Cas systems and play a signific... more ABSTRACTCas1 and Cas2 are highly conserved proteins across CRISPR-Cas systems and play a significant role in protospacer acquisition. Here we study the protospacer (or ps) DNA binding, recognition, and response to cleavage on the protospacer-adjacent-motif complementary sequence or PAMc by Cas1-Cas2, implementing all-atom molecular dynamics simulations. First, we noticed that two active sites of Cas1&1’ bind asymmetrically to two identical PAMc in the simulation. For psDNA containing only one PAMc to be recognized, it is then found that the non-PAMc association site remains destabilized until after the bound PAMc being cleaved. Thus, correlation appears to exist between the two active sites, which can be allosterically mediated by psDNA and Cas2&2’ in bridging. To substantiate such findings, we further simulated Cas1-Cas2 in complex with synthesized psDNA sequences psL and psH, which have been measured with low and high efficiency in acquisition, respectively. Notably, such inter-si...
Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes... more Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes via leucine zipper with its associated partner protein Max to form a heterodimer structure, which then binds target DNA sequences to regulate gene transcription. The regulation depends on by Myc-Max binding to DNA and searching for target sequences via diffusional motions along DNA. Here, we conduct structure-based molecular dynamics (MD) simulations to investigate the diffusion dynamics of the Myc-Max heterodimer along DNA. We found that the heterodimer protein slides on the DNA in a rotation-uncoupled manner in coarse-grained simulations, as its two helical DNA binding basic regions (BRs) alternate between open and closed conformations via inchworm stepping motions. In such motions, the two BRs of the heterodimer step across the DNA strand one by one, with step sizes up about half of a DNA helical pitch length. Atomic MD simulations of the Myc-Max heterodimer in complex with DNA have ...
Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes... more Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes via leucine zipper with its associated partner protein Max to form a heterodimer structure, which then binds target DNA sequences to regulate gene transcription. The regulation depends on by Myc-Max binding to DNA and searching for target sequences via diffusional motions along DNA. Here, we conduct structure-based molecular dynamics (MD) simulations to investigate the diffusion dynamics of the Myc-Max heterodimer along DNA. We found that the heterodimer protein slides on the DNA in a rotation-uncoupled manner in coarse-grained simulations, as its two helical DNA binding basic regions (BRs) alternate between open and closed conformations via inchworm stepping motions. In such motions, the two BRs of the heterodimer step across the DNA strand one by one, with step sizes up about half of a DNA helical pitch length. Atomic MD simulations of the Myc-Max heterodimer in complex with DNA have ...
Transcription factor (TF) target search on genome is highly essential for gene expression and reg... more Transcription 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 refo...
Polymerases select nucleotides according to a template before incorporating them for chemical syn... more Polymerases select nucleotides according to a template before incorporating them for chemical synthesis during gene replication or transcription. Efficient selection to achieve sufficiently high fidelity and speed is essential for polymerase function. Due to multiple kinetic steps detected in a polymerase elongation cycle, there exist multiple selection checkpoints to allow different strategies of fidelity control. In our current work, we examined step-by-step selections in an elongation cycle that have conformational transition rates tuned one at a time, with a controlled differentiation free energy between the right and wrong nucleotides at each checkpoint. The elongation is sustained at non-equilibrium steady state with constant free energy input and heat dissipation. It is found that a selection checkpoint in the later stage of a reaction path has less capability for error reduction. Hence, early selection is essential to achieve an efficient fidelity control. In particular, for...
Rotary sequential hydrolysis of the metabolic machine F1-ATPase is a prominent manifestation of h... more Rotary sequential hydrolysis of the metabolic machine F1-ATPase is a prominent manifestation of high coordination among multiple chemical sites in ring-shaped molecular machines, and it is also functionally essential for F1 to tightly couple chemical reactions and central γ-shaft rotation. High-speed AFM experiments have identified that sequential hydrolysis is maintained in the F1 stator ring even in the absence of the γ-rotor. To explore the origins of intrinsic sequential performance, we computationally investigated essential inter-subunit couplings on the hexameric ring of mitochondrial and bacterial F1. We first reproduced in stochastic Monte Carlo simulations the experimentally determined sequential hydrolysis schemes by kinetically imposing inter-subunit couplings and following subsequent tri-site ATP hydrolysis cycles on the F1 ring. We found that the key couplings to support the sequential hydrolysis are those that accelerate neighbor-site ADP and Pi release upon a certain ...
Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA du... more 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...
Molecular motors are adenosine tri-phosphate (ATP) hydrolysis-driven, cellular proteins responsib... more Molecular motors are adenosine tri-phosphate (ATP) hydrolysis-driven, cellular proteins responsible for a wide variety of different tasks, such as transport, energy metabolism, and DNA processing. Their operation cycle spans a wide range of length and time scales, from the localized and fast chemical reaction in the catalytic site(s) to the large scale and much slower conformational motions involved in the motors' physiological function. From a computational point of view, this means that currently there exists no single approach capable of capturing the whole spectrum of events during molecular motor function. In the present review, we show for PcrA helicase, a molecular motor involved in the unwinding of double-stranded DNA, how a combination of computational approaches can be used to examine PcrA's function in its entirety as well as in detail. Combined quantum mechanical/molecular mechanical simulations are used to study the catalytic ATP hydrolysis event and its coupling to protein conformational changes. Molecular dynamics simulations then provide a means of studying overall PcrA function on a nanosecond time scale. Finally, to reach physiologically relevant time scales, i.e., milliseconds, stochastic simulations are employed. We show that by combining the three stated approaches one can obtain insight into PcrA helicase function.
Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucle... more 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 an...
How do mammalian cells that share the same genome exist in notably distinct phenotypes, exhibitin... more How do mammalian cells that share the same genome exist in notably distinct phenotypes, exhibiting differences in morphology, gene expression patterns, and epigenetic chromatin statuses? Furthermore how do cells of different phenotypes differentiate reproducibly from a single fertilized egg? These are fundamental problems in developmental biology. Epigenetic histone modifications play an important role in the maintenance of different cell phenotypes. The exact molecular mechanism for inheritance of the modification patterns over cell generations remains elusive. The complexity comes partly from the number of molecular species and the broad time scales involved. In recent years mathematical modeling has made significant contributions on elucidating the molecular mechanisms of DNA methylation and histone Book chapter in Epigenetic Technological Applications (Elsevier), in press 2 covalent modification inheritance. We will pedagogically introduce the typical procedure and some technical details of performing a mathematical modeling study, and discuss future developments.
Nucleotide selection is essential for fidelity control in gene replication and transcription. Rec... more Nucleotide selection is essential for fidelity control in gene replication and transcription. Recent work on T7 RNA polymerase suggested that a small posttranslocation free energy bias stabilizes Tyr(639) in the active site to aid nucleotide selection. However, it was not clear exactly how Tyr(639) assists the selection. Here we report a molecular-dynamics simulation study revealing atomistic detail of this critical selectivity. The study shows first that Tyr(639) blocks the active site at posttranslocation by marginally stacking to the end basepair of the DNA-RNA hybrid. The study then demonstrates that at the nucleotide preinsertion state, a cognate RNA nucleotide does not affect the local Tyr(639) stabilization, whereas a noncognate nucleotide substantially stabilizes Tyr(639) so that Tyr(639) keeps blocking the active site. As a result, further nucleotide insertion into the active site, which requires moving Tyr(639) out of the site, would be hindered for the noncognate nucleoti...
The RNA polymerase II elongation is central in eukaryotic transcription. Although multiple interm... more The RNA polymerase II elongation is central in eukaryotic transcription. Although multiple intermediates of the elongation complex have been identified, the dynamical mechanisms remain elusive or controversial. Here we build a structure-based kinetic model of a full elongation cycle of polymerase II, taking into account transition rates and conformational changes characterized from both single molecule experimental studies and computational simulations at atomistic scale. Our model suggests a force-dependent slow transition detected in the single molecule experiments corresponds to an essential conformational change of a trigger loop (TL) opening prior to the polymerase translocation. The analyses on mutant study of E1103G and on potential sequence effects of the translocation substantiate this proposal. Our model also investigates another slow transition detected in the transcription elongation cycle which is independent of mechanical force. If this force-independent slow transitio...
The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage phi29 virus ... more The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage phi29 virus capsid. On the basis of the results of single-molecule experimental studies, we propose a push and roll mechanism to explain how the packaging motor translocates the DNA in bursts of four 2.5 bp power strokes, while rotating the DNA. In this mechanism, each power stroke accompanies P(i) release after ATP hydrolysis. Since the high-resolution structure of the gp16 motor is not available, we borrowed characterized features from the P4 RNA packaging motor in bacteriophage phi12. For each power stroke, a lumenal lever from a single subunit is electrostatically steered to the DNA backbone. The lever then pushes sterically, orthogonal to the backbone axis, such that the right-handed DNA helix is translocated and rotated in a left-handed direction. The electrostatic association allows tight coupling between the lever and the DNA and prevents DNA from slipping back. The lever affinity for DNA de...
The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage phi29 virus ... more The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage phi29 virus capsid. On the basis of the results of single-molecule experimental studies, we propose a push and roll mechanism to explain how the packaging motor translocates the DNA in bursts of four 2.5 bp power strokes, while rotating the DNA. In this mechanism, each power stroke accompanies P(i) release after ATP hydrolysis. Since the high-resolution structure of the gp16 motor is not available, we borrowed characterized features from the P4 RNA packaging motor in bacteriophage phi12. For each power stroke, a lumenal lever from a single subunit is electrostatically steered to the DNA backbone. The lever then pushes sterically, orthogonal to the backbone axis, such that the right-handed DNA helix is translocated and rotated in a left-handed direction. The electrostatic association allows tight coupling between the lever and the DNA and prevents DNA from slipping back. The lever affinity for DNA de...
During stress, cardiac function is enhanced via b-adrenergic stimulation. Phosphorylation of card... more During stress, cardiac function is enhanced via b-adrenergic stimulation. Phosphorylation of cardiac ryanodine receptors (RyR2) has been proposed to lead to increased Ca 2þ release from the sarcoplasmic reticulum (SR), but experimental findings are controversial. Using cardiomyocytes lacking the PKA phosphorylation site Ser2808 on RyR2 we previously observed that this site contributes to improved spatio-temporal synchronization of Ca 2þ release during EC-coupling under b-adrenergic stimulation, presumably by a Ca 2þ sensing mechanism inside the SR. Here, we further tested the hypothesis that Ser2808 phosphorylation modulates RyR2 function via its dependence on [Ca 2þ ] SR. Cardiomyocytes from control and S2808A mice were loaded with fluo-3-AM and Ca 2þ was imaged with confocal line-scans. Ca 2þ waves were induced by increasing external [Ca 2þ ] to 10mM and analyzed in control and during b-stimulation with isoproterenol (1mM). In control conditions Ca 2þ transients during a wave showed a comparable amplitude and time course. However, wave speed was reduced by 25% in S2808A cells, possibly related to a lack of background phosphorylation at this site. While ISO increased wave speed by 15% in control cells (from 98 to 113mm/s), it remained unaffected in S2808A cells (from 73 to 75mm/s). This situation could be reproduced in control cells preincubated with the PKA inhibitor H89 or the CaMKII inhibitor KN93, indicating a complex regulation of CICR that is pharmacologically difficult to dissect in intact cells. Taken together our findings in S2808A myocytes reveal a lack of Ca 2þ release synchronization (via an intra-SR Ca 2þ sensing mechanism) and an ablation of Ca 2þ wave speed modulation (presumably by the same mechanism) by b-adrenergic stimulation. Thus, Ser2808 phosphorylation may change the sensitivity of the RyR2 for intra-SR Ca 2þ. Hyperphosphorylation of Ser2808, as in failing hearts, may thereby render RyR2 hypersensitive and contribute to increased Ca 2þ leak.
The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA ... more The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA to RNA in the absence of additional protein factors. In this work, we present a model of T7 RNAP translocation during elongation. Based on structural information and experimental data from single-molecule force measurements, we show that a small component of facilitated translocation or power stroke coexists with the Brownian-ratchet-driven motions, and plays a crucial role in nucleotide selection at pre-insertion. The facilitated translocation is carried out by the conserved Tyr 639 that moves its side chain into the active site, pushing aside the 3 0-end of the RNA, and forming a locally stabilized post-translocation intermediate. Pre-insertion of an incoming nucleotide into this stabilized intermediate state ensures that Tyr 639 closely participates in selecting correct nucleotides. A similar translocation mechanism has been suggested for multi-subunit RNAPs involving the bridge-helix bending. Nevertheless, the bent bridge-helix sterically prohibits nucleotide binding in the post-transolocation intermediate analog; moreover, the analog is not stabilized unless an inhibitory protein factor binds to the enzyme. Using our scheme, we also compared the efficiencies of different strategies for nucleotide selection, and examined effects of facilitated translocation on forward tracking.
PcrA helicase from Bacillus stearothermophilus is one of the smallest motor proteins structurally... more PcrA helicase from Bacillus stearothermophilus is one of the smallest motor proteins structurally known in full atomic detail. It translocates progressively from the 39 end to the 59 end of single-stranded DNA utilizing the free energy from ATP hydrolysis. The similarities in structure and reaction pathway between PcrA helicase and F1-ATPase suggest a similar mechanochemical mechanism at work in both systems. Previous studies of PcrA translocation demonstrated a domain stepping mechanism in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of two translocation domains is synchronized with alternating mobilities of the individual domains such that PcrA moves unidirectionally along single-stranded DNA. To substantiate this translocation mechanism, this study applies molecular dynamics simulations, elastic network theory, and multiple sequence alignment to analyze the system. The analysis provides further evidence that directional translocation of PcrA is regulated allosterically through synchronization of ATP hydrolysis and domain mobilities. We identify a set of essential residues coevolutionarily coupled in related helicases that should be involved in the allosteric regulation of these motor proteins.
DNA helicases are ubiquitous molecular motors involved in cellular DNA metabolism. They move alon... more DNA helicases are ubiquitous molecular motors involved in cellular DNA metabolism. They move along singlestranded DNA (ssDNA) and separate duplex DNA into its component strands, utilizing the free energy from ATP hydrolysis. The PcrA helicase from Bacillus stearothermophilus translocates as a monomer progressively from the 39 end to the 59 end of ssDNA and is one of the smallest motor proteins structurally known in full atomic detail. Using high-resolution crystal structures of the PcrA-DNA complex, we performed nanosecond molecular dynamics simulations and derived potential energy profiles governing individual domain movement of the PcrA helicase along ssDNA. Based on these profiles, the millisecond translocation of the helicase along ssDNA was described through Langevin dynamics. The calculations support a domain stepping mechanism of PcrA helicase, in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of domains 2A and 1A are synchronized with alternating mobilities of the individual domains in such a fashion that PcrA moves unidirectionally along ssDNA. By combining short timescale (nanoseconds) molecular dynamics and long timescale (milliseconds) stochasticdynamics descriptions, our study suggests a structure-based mechanism of the ATP-powered unidirectional movement of PcrA helicase.
Homologous recombination plays a key role in the restart of stalled replication forks and in the ... more Homologous recombination plays a key role in the restart of stalled replication forks and in the generation of genetic diversity. During this process, two homologous DNA molecules undergo strand exchange to form a four-way DNA (Holliday) junction. In the presence of metal ions, the Holliday junction folds into the stacked-X structure that has two alternative conformers. Experiments have revealed the spontaneous transitions between these conformers, but their detailed pathways are not known. Here, we report a series of molecular dynamics simulations of the Holliday junction at physiological and elevated (400 K) temperatures. The simulations reveal new tetrahedral intermediates and suggest a schematic framework for conformer transitions. The tetrahedral intermediates bear resemblance to the junction conformation in complex with a junction-resolving enzyme, T7 endonuclease I, and indeed, one intermediate forms a stable complex with the enzyme as demonstrated in one simulation. We also describe free energy minima for various states of the Holliday junction system, which arise during conformer transitions. The results show that magnesium ions stabilize the stacked-X form and destabilize the open and tetrahedral intermediates. Overall, our study provides a detailed dynamic model of the Holliday junction undergoing a conformer transition.
ABSTRACTCas1 and Cas2 are highly conserved proteins across CRISPR-Cas systems and play a signific... more ABSTRACTCas1 and Cas2 are highly conserved proteins across CRISPR-Cas systems and play a significant role in protospacer acquisition. Here we study the protospacer (or ps) DNA binding, recognition, and response to cleavage on the protospacer-adjacent-motif complementary sequence or PAMc by Cas1-Cas2, implementing all-atom molecular dynamics simulations. First, we noticed that two active sites of Cas1&1’ bind asymmetrically to two identical PAMc in the simulation. For psDNA containing only one PAMc to be recognized, it is then found that the non-PAMc association site remains destabilized until after the bound PAMc being cleaved. Thus, correlation appears to exist between the two active sites, which can be allosterically mediated by psDNA and Cas2&2’ in bridging. To substantiate such findings, we further simulated Cas1-Cas2 in complex with synthesized psDNA sequences psL and psH, which have been measured with low and high efficiency in acquisition, respectively. Notably, such inter-si...
Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes... more Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes via leucine zipper with its associated partner protein Max to form a heterodimer structure, which then binds target DNA sequences to regulate gene transcription. The regulation depends on by Myc-Max binding to DNA and searching for target sequences via diffusional motions along DNA. Here, we conduct structure-based molecular dynamics (MD) simulations to investigate the diffusion dynamics of the Myc-Max heterodimer along DNA. We found that the heterodimer protein slides on the DNA in a rotation-uncoupled manner in coarse-grained simulations, as its two helical DNA binding basic regions (BRs) alternate between open and closed conformations via inchworm stepping motions. In such motions, the two BRs of the heterodimer step across the DNA strand one by one, with step sizes up about half of a DNA helical pitch length. Atomic MD simulations of the Myc-Max heterodimer in complex with DNA have ...
Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes... more Oncogenic protein Myc serves as a transcription factor to control cell metabolisms. Myc dimerizes via leucine zipper with its associated partner protein Max to form a heterodimer structure, which then binds target DNA sequences to regulate gene transcription. The regulation depends on by Myc-Max binding to DNA and searching for target sequences via diffusional motions along DNA. Here, we conduct structure-based molecular dynamics (MD) simulations to investigate the diffusion dynamics of the Myc-Max heterodimer along DNA. We found that the heterodimer protein slides on the DNA in a rotation-uncoupled manner in coarse-grained simulations, as its two helical DNA binding basic regions (BRs) alternate between open and closed conformations via inchworm stepping motions. In such motions, the two BRs of the heterodimer step across the DNA strand one by one, with step sizes up about half of a DNA helical pitch length. Atomic MD simulations of the Myc-Max heterodimer in complex with DNA have ...
Transcription factor (TF) target search on genome is highly essential for gene expression and reg... more Transcription 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 refo...
Polymerases select nucleotides according to a template before incorporating them for chemical syn... more Polymerases select nucleotides according to a template before incorporating them for chemical synthesis during gene replication or transcription. Efficient selection to achieve sufficiently high fidelity and speed is essential for polymerase function. Due to multiple kinetic steps detected in a polymerase elongation cycle, there exist multiple selection checkpoints to allow different strategies of fidelity control. In our current work, we examined step-by-step selections in an elongation cycle that have conformational transition rates tuned one at a time, with a controlled differentiation free energy between the right and wrong nucleotides at each checkpoint. The elongation is sustained at non-equilibrium steady state with constant free energy input and heat dissipation. It is found that a selection checkpoint in the later stage of a reaction path has less capability for error reduction. Hence, early selection is essential to achieve an efficient fidelity control. In particular, for...
Rotary sequential hydrolysis of the metabolic machine F1-ATPase is a prominent manifestation of h... more Rotary sequential hydrolysis of the metabolic machine F1-ATPase is a prominent manifestation of high coordination among multiple chemical sites in ring-shaped molecular machines, and it is also functionally essential for F1 to tightly couple chemical reactions and central γ-shaft rotation. High-speed AFM experiments have identified that sequential hydrolysis is maintained in the F1 stator ring even in the absence of the γ-rotor. To explore the origins of intrinsic sequential performance, we computationally investigated essential inter-subunit couplings on the hexameric ring of mitochondrial and bacterial F1. We first reproduced in stochastic Monte Carlo simulations the experimentally determined sequential hydrolysis schemes by kinetically imposing inter-subunit couplings and following subsequent tri-site ATP hydrolysis cycles on the F1 ring. We found that the key couplings to support the sequential hydrolysis are those that accelerate neighbor-site ADP and Pi release upon a certain ...
Here, we studied the complete process of a viral T7 RNA polymerase (RNAP) translocation on DNA du... more 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...
Molecular motors are adenosine tri-phosphate (ATP) hydrolysis-driven, cellular proteins responsib... more Molecular motors are adenosine tri-phosphate (ATP) hydrolysis-driven, cellular proteins responsible for a wide variety of different tasks, such as transport, energy metabolism, and DNA processing. Their operation cycle spans a wide range of length and time scales, from the localized and fast chemical reaction in the catalytic site(s) to the large scale and much slower conformational motions involved in the motors' physiological function. From a computational point of view, this means that currently there exists no single approach capable of capturing the whole spectrum of events during molecular motor function. In the present review, we show for PcrA helicase, a molecular motor involved in the unwinding of double-stranded DNA, how a combination of computational approaches can be used to examine PcrA's function in its entirety as well as in detail. Combined quantum mechanical/molecular mechanical simulations are used to study the catalytic ATP hydrolysis event and its coupling to protein conformational changes. Molecular dynamics simulations then provide a means of studying overall PcrA function on a nanosecond time scale. Finally, to reach physiologically relevant time scales, i.e., milliseconds, stochastic simulations are employed. We show that by combining the three stated approaches one can obtain insight into PcrA helicase function.
Pyrophosphate ion (PPi) release during transcription elongation is a signature step in each nucle... more 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 an...
How do mammalian cells that share the same genome exist in notably distinct phenotypes, exhibitin... more How do mammalian cells that share the same genome exist in notably distinct phenotypes, exhibiting differences in morphology, gene expression patterns, and epigenetic chromatin statuses? Furthermore how do cells of different phenotypes differentiate reproducibly from a single fertilized egg? These are fundamental problems in developmental biology. Epigenetic histone modifications play an important role in the maintenance of different cell phenotypes. The exact molecular mechanism for inheritance of the modification patterns over cell generations remains elusive. The complexity comes partly from the number of molecular species and the broad time scales involved. In recent years mathematical modeling has made significant contributions on elucidating the molecular mechanisms of DNA methylation and histone Book chapter in Epigenetic Technological Applications (Elsevier), in press 2 covalent modification inheritance. We will pedagogically introduce the typical procedure and some technical details of performing a mathematical modeling study, and discuss future developments.
Nucleotide selection is essential for fidelity control in gene replication and transcription. Rec... more Nucleotide selection is essential for fidelity control in gene replication and transcription. Recent work on T7 RNA polymerase suggested that a small posttranslocation free energy bias stabilizes Tyr(639) in the active site to aid nucleotide selection. However, it was not clear exactly how Tyr(639) assists the selection. Here we report a molecular-dynamics simulation study revealing atomistic detail of this critical selectivity. The study shows first that Tyr(639) blocks the active site at posttranslocation by marginally stacking to the end basepair of the DNA-RNA hybrid. The study then demonstrates that at the nucleotide preinsertion state, a cognate RNA nucleotide does not affect the local Tyr(639) stabilization, whereas a noncognate nucleotide substantially stabilizes Tyr(639) so that Tyr(639) keeps blocking the active site. As a result, further nucleotide insertion into the active site, which requires moving Tyr(639) out of the site, would be hindered for the noncognate nucleoti...
The RNA polymerase II elongation is central in eukaryotic transcription. Although multiple interm... more The RNA polymerase II elongation is central in eukaryotic transcription. Although multiple intermediates of the elongation complex have been identified, the dynamical mechanisms remain elusive or controversial. Here we build a structure-based kinetic model of a full elongation cycle of polymerase II, taking into account transition rates and conformational changes characterized from both single molecule experimental studies and computational simulations at atomistic scale. Our model suggests a force-dependent slow transition detected in the single molecule experiments corresponds to an essential conformational change of a trigger loop (TL) opening prior to the polymerase translocation. The analyses on mutant study of E1103G and on potential sequence effects of the translocation substantiate this proposal. Our model also investigates another slow transition detected in the transcription elongation cycle which is independent of mechanical force. If this force-independent slow transitio...
The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage phi29 virus ... more The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage phi29 virus capsid. On the basis of the results of single-molecule experimental studies, we propose a push and roll mechanism to explain how the packaging motor translocates the DNA in bursts of four 2.5 bp power strokes, while rotating the DNA. In this mechanism, each power stroke accompanies P(i) release after ATP hydrolysis. Since the high-resolution structure of the gp16 motor is not available, we borrowed characterized features from the P4 RNA packaging motor in bacteriophage phi12. For each power stroke, a lumenal lever from a single subunit is electrostatically steered to the DNA backbone. The lever then pushes sterically, orthogonal to the backbone axis, such that the right-handed DNA helix is translocated and rotated in a left-handed direction. The electrostatic association allows tight coupling between the lever and the DNA and prevents DNA from slipping back. The lever affinity for DNA de...
The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage phi29 virus ... more The pentameric ATPase motor gp16 packages double-stranded DNA into the bacteriophage phi29 virus capsid. On the basis of the results of single-molecule experimental studies, we propose a push and roll mechanism to explain how the packaging motor translocates the DNA in bursts of four 2.5 bp power strokes, while rotating the DNA. In this mechanism, each power stroke accompanies P(i) release after ATP hydrolysis. Since the high-resolution structure of the gp16 motor is not available, we borrowed characterized features from the P4 RNA packaging motor in bacteriophage phi12. For each power stroke, a lumenal lever from a single subunit is electrostatically steered to the DNA backbone. The lever then pushes sterically, orthogonal to the backbone axis, such that the right-handed DNA helix is translocated and rotated in a left-handed direction. The electrostatic association allows tight coupling between the lever and the DNA and prevents DNA from slipping back. The lever affinity for DNA de...
During stress, cardiac function is enhanced via b-adrenergic stimulation. Phosphorylation of card... more During stress, cardiac function is enhanced via b-adrenergic stimulation. Phosphorylation of cardiac ryanodine receptors (RyR2) has been proposed to lead to increased Ca 2þ release from the sarcoplasmic reticulum (SR), but experimental findings are controversial. Using cardiomyocytes lacking the PKA phosphorylation site Ser2808 on RyR2 we previously observed that this site contributes to improved spatio-temporal synchronization of Ca 2þ release during EC-coupling under b-adrenergic stimulation, presumably by a Ca 2þ sensing mechanism inside the SR. Here, we further tested the hypothesis that Ser2808 phosphorylation modulates RyR2 function via its dependence on [Ca 2þ ] SR. Cardiomyocytes from control and S2808A mice were loaded with fluo-3-AM and Ca 2þ was imaged with confocal line-scans. Ca 2þ waves were induced by increasing external [Ca 2þ ] to 10mM and analyzed in control and during b-stimulation with isoproterenol (1mM). In control conditions Ca 2þ transients during a wave showed a comparable amplitude and time course. However, wave speed was reduced by 25% in S2808A cells, possibly related to a lack of background phosphorylation at this site. While ISO increased wave speed by 15% in control cells (from 98 to 113mm/s), it remained unaffected in S2808A cells (from 73 to 75mm/s). This situation could be reproduced in control cells preincubated with the PKA inhibitor H89 or the CaMKII inhibitor KN93, indicating a complex regulation of CICR that is pharmacologically difficult to dissect in intact cells. Taken together our findings in S2808A myocytes reveal a lack of Ca 2þ release synchronization (via an intra-SR Ca 2þ sensing mechanism) and an ablation of Ca 2þ wave speed modulation (presumably by the same mechanism) by b-adrenergic stimulation. Thus, Ser2808 phosphorylation may change the sensitivity of the RyR2 for intra-SR Ca 2þ. Hyperphosphorylation of Ser2808, as in failing hearts, may thereby render RyR2 hypersensitive and contribute to increased Ca 2þ leak.
The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA ... more The RNA polymerase (RNAP) of bacteriophage T7 is a single subunit enzyme that can transcribe DNA to RNA in the absence of additional protein factors. In this work, we present a model of T7 RNAP translocation during elongation. Based on structural information and experimental data from single-molecule force measurements, we show that a small component of facilitated translocation or power stroke coexists with the Brownian-ratchet-driven motions, and plays a crucial role in nucleotide selection at pre-insertion. The facilitated translocation is carried out by the conserved Tyr 639 that moves its side chain into the active site, pushing aside the 3 0-end of the RNA, and forming a locally stabilized post-translocation intermediate. Pre-insertion of an incoming nucleotide into this stabilized intermediate state ensures that Tyr 639 closely participates in selecting correct nucleotides. A similar translocation mechanism has been suggested for multi-subunit RNAPs involving the bridge-helix bending. Nevertheless, the bent bridge-helix sterically prohibits nucleotide binding in the post-transolocation intermediate analog; moreover, the analog is not stabilized unless an inhibitory protein factor binds to the enzyme. Using our scheme, we also compared the efficiencies of different strategies for nucleotide selection, and examined effects of facilitated translocation on forward tracking.
PcrA helicase from Bacillus stearothermophilus is one of the smallest motor proteins structurally... more PcrA helicase from Bacillus stearothermophilus is one of the smallest motor proteins structurally known in full atomic detail. It translocates progressively from the 39 end to the 59 end of single-stranded DNA utilizing the free energy from ATP hydrolysis. The similarities in structure and reaction pathway between PcrA helicase and F1-ATPase suggest a similar mechanochemical mechanism at work in both systems. Previous studies of PcrA translocation demonstrated a domain stepping mechanism in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of two translocation domains is synchronized with alternating mobilities of the individual domains such that PcrA moves unidirectionally along single-stranded DNA. To substantiate this translocation mechanism, this study applies molecular dynamics simulations, elastic network theory, and multiple sequence alignment to analyze the system. The analysis provides further evidence that directional translocation of PcrA is regulated allosterically through synchronization of ATP hydrolysis and domain mobilities. We identify a set of essential residues coevolutionarily coupled in related helicases that should be involved in the allosteric regulation of these motor proteins.
DNA helicases are ubiquitous molecular motors involved in cellular DNA metabolism. They move alon... more DNA helicases are ubiquitous molecular motors involved in cellular DNA metabolism. They move along singlestranded DNA (ssDNA) and separate duplex DNA into its component strands, utilizing the free energy from ATP hydrolysis. The PcrA helicase from Bacillus stearothermophilus translocates as a monomer progressively from the 39 end to the 59 end of ssDNA and is one of the smallest motor proteins structurally known in full atomic detail. Using high-resolution crystal structures of the PcrA-DNA complex, we performed nanosecond molecular dynamics simulations and derived potential energy profiles governing individual domain movement of the PcrA helicase along ssDNA. Based on these profiles, the millisecond translocation of the helicase along ssDNA was described through Langevin dynamics. The calculations support a domain stepping mechanism of PcrA helicase, in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of domains 2A and 1A are synchronized with alternating mobilities of the individual domains in such a fashion that PcrA moves unidirectionally along ssDNA. By combining short timescale (nanoseconds) molecular dynamics and long timescale (milliseconds) stochasticdynamics descriptions, our study suggests a structure-based mechanism of the ATP-powered unidirectional movement of PcrA helicase.
Packaging the genome of a virus into its capsid is a crucial step in viral assembly. The genome o... more Packaging the genome of a virus into its capsid is a crucial step in viral assembly. The genome of bacteriophage φ29 consists of a linear doublestranded DNA (dsDNA) of about 19,000 base pairs (bps). Packaging a dsDNA this long results in a near-crystalline state inside the ~50 nm length capsid and requires a great deal of energy. The feat is performed by a multimeric molecular motor that derives its energy from ATP hydrolysis and generates forces more than 60 pN . Experimental studies on the φ29 packaging motor have been carried out through single-molecule manipulation techniques using optical tweezers . The DNA packaging proceeds in bursts of four 2.5-bp translocation power strokes upon Pi releases. The translocation is also accompanied by the DNA rotation. From the data we have constructed a mechanochemical framework to explain how this motor packages DNA. The model is built around 'push-and-roll' mechanism that suggests how the motor subunits interact with the DNA and how the DNA passes through the motor ring. We also propose how the five subunits are coordinated around the ring. Our model provides a new perspective on how multimeric ATPases transport nucleic-acids, and it may be applied to other ring motors.
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Papers by Jin Yu