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Homologs of the mutagenic Escherichia coli DNA polymerase V (pol V) are encoded by numerous pathogens and mobile elements. We have used Rum pol (RumA′2B), from the integrative conjugative element (ICE), R391, as a model mobile element-encoded polymerase (MEPol). The highly mutagenic Rum pol is transferred horizontally into a variety of recipient cells, including many pathogens. Moving between species, it is unclear if Rum pol can function on its own or requires activation by host factors. Here, we show that Rum pol biochemical activity requires the formation of a physical mutasomal complex, Rum Mut, containing RumA′2B-RecA-ATP, with RecA being donated by each recipient bacteria. For R391, Rum Mut specific activities in vitro and mutagenesis rates in vivo depend on the phylogenetic distance of host-cell RecA from E. coli RecA. Rum pol is a highly conserved and effective mobile catalyst of rapid evolution, with the potential to generate a broad mutational landscape that could serve to ensure bacterial adaptation in antibiotic-rich environments leading to the establishment of antibiotic resistance.

Trinucleotide repeat expansion disorders are associated with the overexpansion of (CNG) repeats on the genome. Messenger RNA transcripts of sequences with greater than 60-100 (CNG) tandem units have been implicated in trinucleotide repeat expansion disorder pathogenesis. In this work, we develop a diagrammatic theory to study the structural diversity of these (CNG)n RNA sequences. Representing structural elements on the chain's conformation by a set of graphs and employing elementary diagrammatic methods, we have formulated a renormalization procedure to re-sum these graphs and arrive at a closed-form expression for the ensemble partition function. With a simple approximation for the renormalization and applied to extended (CNG)n sequences, this theory can comprehensively capture an infinite set of conformations with any number and any combination of duplexes, hairpins, multiway junctions, and quadruplexes. To quantify the diversity of different (CNG)n ensembles, the analytical equations derived from the diagrammatic theory were solved numerically to derive equilibrium estimates for the secondary structural contents of the chains. The results suggest that the structural ensembles of (CNG)n repeat sequence with n ∼60 are surprisingly diverse, and the distribution is sensitive to the ability of the N nucleotide to make noncanonical pairs and whether the (CNG)n sequence can sustain stable quadruplexes. The results show how perturbations in the form of biases on the stabilities of the various structural motifs, duplexes, junctions, helices, and quadruplexes could affect the secondary structures of the chains and how these structures may switch when they are perturbed.

During the COVID-19 pandemic, an at-home laboratory program was created and implemented for a section of the general chemistry course at the University of Southern California. The experiments were designed to only utilize safe household items and no special equipment. These laboratory activities, spanning over 4 weeks, focused on concepts usually covered in the final one-third of our second-semester chemistry laboratory, including pH, acid–base titrations, buffers, solubility, phase equilibria, and thermodynamics. In this article, we describe the design of the laboratories and our experience with this experiment, while also providing an assessment on how similar activities could be integrated profitably into a regular general chemistry course.

Performing full-resolution atomistic simulations of nucleic acid folding has remained a challenge for biomolecular modeling. Understanding how nucleic acids fold and how they transition between different folded structures as they unfold and refold has important implications for biology. This paper reports a theoretical model and computer simulation of the ab initio folding of DNA inverted repeat sequences. The formulation is based on an all-atom conformational model of the sugar-phosphate backbone via chain closure, and it incorporates three major molecular-level driving forces—base stacking, counterion-induced backbone self-interactions, and base pairing—via separate analytical theories designed to capture and reproduce the effects of the solvent without requiring explicit water and ions in the simulation. To accelerate computational throughput, a mixed numerical/analytical algorithm for the calculation of the backbone conformational volume is incorporated into the Monte Carlo simulation, and special stochastic sampling techniques were employed to achieve the computational efficiency needed to fold nucleic acids from scratch. This paper describes implementation details, benchmark results, and the advantages and technical challenges with this approach.

Activation-induced deoxycytidine deaminase (AID) is a key enzyme in the human immune system. AID binds to and catalyzes random point mutations on the immunoglobulin (Ig) gene, leading to diversification of the Ig gene sequence by random walk motions, scanning for cytidines and turning them to uracils. The mutation patterns deposited by AID on its substrate DNA sequences can be interpreted as random binary words, and the information content of this stochastically-generated library of mutated DNA sequences can be measured by its entropy. In this paper, we derive an analytical formula for this entropy and show that the stochastic scanning + catalytic dynamics of AID is controlled by a characteristic length that depends on the diffusion coefficient of AID and the catalytic rate. Experiments showed that the deamination rates have a sequence context dependence, where mutations are generated at higher intensities on DNA sequences with higher densities of mutable sites. We derive an isomorphism between this classical system and a quantum mechanical model and use this isomorphism to explain why AID appears to focus its scanning on regions with higher concentrations of deaminable sites. Using path integral Monte Carlo simulations of the quantum isomorphic system, we demonstrate how AID’s scanning indeed depends on the context of the DNA sequence and how this affects the entropy of the library of generated mutant clones. Examining detailed features in the entropy of the experimentally-generated clone library, we provide clear evidence that the random walk of AID on its substrate DNA is focused near hot spots. The model calculations applied to the experimental data show that the observed per-site mutation frequencies display similar contextual dependences as observed in the experiments, in which hot motifs are located adjacent to several different types of hot and cold motifs.

Base pairing plays a pivotal role in DNA functions and replication fidelity. But while the complementarity between Watson-Crick matched bases is generally believed to arise from the different number of hydrogen bonds in G|C pairs versus A|T, the energetics of these interactions are heavily renormalized by the aqueous solvent. Employing large-scale Monte Carlo simulations, we have extracted the solvent contribution to the free energy for canonical and some noncanonical and stacked base pairs. For all of them, the solvent’s contribution to the base pairing free energy is exclusively destabilizing. While the direct hydrogen bonding interactions in the G|C pair is much stronger than A|T, the thermodynamic resistance produced by the solvent also pushes back much stronger against G|C compared to A|T, generating an only ~1 kcal/mol free energy difference between them. We have profiled the density of water molecules in the solvent adjacent to the bases and observed a “freezing” behavior where waters are recruited into the gap between the bases to compensate for the unsatisfied hydrogen bonds between them. A very small number of water molecules that are associated with the Watson-Crick donor/acceptor atoms turn out to be responsible for the majority of solvent’s thermodynamic resistance to base pairing. The absence or presence of these near-field waters can be used to enhance fidelity during DNA replication.TOC Graphic

Ultrahigh-throughput conformational sampling of biopolymers like nucleic acids are most effectively carried out without explicit solvents, but the physical origins of almost all inter- and intramolecular interactions controlling nucleic acid structures are rooted in water. Single-stranded (ss) DNAs or RNAs in water are characterized by ensembles of diverse conformations. To properly capture solvent-induced nucleobase stacking interactions in an otherwise solvent-free Monte Carlo algorithm, theoretical models are developed here to describe the solvent entropy and dispersion terms in base stacking free energies. To validate these models, equilibrium ensembles of ss (dA) n and (dT) n sequences ( n = 30, 40, and 50) were simulated, and they quantitatively reproduced experimental small-angle X-ray scattering (SAXS) data. Simulated dA ensembles show substantial stacking. While less prevalent, stacking in dT chains is not negligible. Analysis of SAXS profiles suggests that excess features between wavevector 0.03 and 0.18 A-1 correlate with stacking, and stacking in dA versus dT chains is chain length-dependent, where (dT)30 and (dA)30 chains have more similar structures, but longer dA chains show more stacking over dT. The average stack length in ss-dA chains is 5-10 nucleotides, yielding an estimate for the overall A|A stacking free energy at ∼1 kcal/mol.

Trinucleotide repeat expansion disorders (TRED) are associated with the overexpansion of (CNG) repeats on the genome. mRNA transcripts of sequences with greater than 60 to 100 (CNG) tandem units have been implicated in TRED pathogenesis. In this paper, we develop a diagrammatic theory to study the structural diversity of these (CNG)n RNA sequences. Representing structural elements on the chain’s conformation by a set of graphs and employing elementary diagrammatic methods, we have formulated a renormalization procedure to resum these graphs and arrive at a closed-form expression for the ensemble partition function. With a simple approximation for the renormalization and applied to extended (CNG)n sequences, this theory can comprehensively capture an infinite set of conformations with any number and any combination of duplexes, hairpins and 2-way junctions. To quantify the diversity of different (CNG)n ensembles, the analytical equations derived from the diagrammatic theory were solved numerically to derive equilibrium estimates for the secondary structural contents of the chains. The results suggest that the structural ensembles of (CNG)n repeat sequence with n ~ 60 are surprisingly diverse, and they are dominated largely by open segments, with only a small fraction of the nucleotides forming base pairs. At the same time, the variance in the secondary-structural contents on the chains is also quite large, indicating that their structures can undergo strong equilibrium fluctuations and are expected to be rather suspectable to perturbations.STATEMENT OF SIGNIFICANCETrinucleotide repeat expansion disorders (TRED) are associated with the overexpansion of (CNG) repeats on the genome. mRNA transcripts of sequences with critical length greater than 60 to 100 (CNG) tandem units have been implicated in TRED pathogenesis, though their structures remain poorly characterized. Conventional view has tacitly assumed that conformations with maximal C:G base pairing dominate at equilibrium, but here we demonstrate that (CNG) repeat sequences are characterized by diverse ensembles of structurally heterogeneous folds and with a large variance of secondary structural contents. These ensembles of structures also undergo strong equilibrium fluctuations, rendering them rather susceptible to perturbations. These results were based on a novel diagrammatic approach to the ensemble partition function.

Turning a linear sugar into its cyclized form is an essential skill in biochemistry. A simple paper model is described that can be used by any student to transform a carbohydrate in its Fischer projection and correctly cyclize it into its Haworth form. The model can also be used to illustrate several key aspects of the Fischer projection of which many students are typically unaware.

Polyaromatic dye molecules employed in photovoltaic and electronic applications are often processed in organic solvents. The aggregation of these dyes is key to their applications, but a fundamental molecular understanding of how the solvent environment controls the stacking of polyaromatics is unclear. This study reports initial results from Monte Carlo simulations of how various acene molecule dimers stack when they are dissolved in different solvents. Free energies computed using full dispersion interactions versus those with sterics only suggest that solvent entropy alone accounts for the majority of the stacking free energy in solvents with compact molecular geometries such as carbon tetrachloride. However, in contrast with carbon tetrachloride, we also observe significant variations in the stacking free energies of naphthalene, anthracene, and tetracene across other solvents such as toluene and cyclohexane. The weak attractive dispersion interactions between the acene solutes and planar and near-pla...

Base stacking is a key determinant of nucleic acid structures, but the precise origin of the thermodynamic driving force behind the stacking of nucleobases remains open. The rather mild stacking free energy measured experimentally, roughly a kcal/mol depending on the identity of the bases, is physiologically significant because while base stacking confers stability to the genome in its double helix form, the duplex also has to be unwound in order to be replicated or transcribed. A stacking free energy that is either too high or too low will over- or understabilize the genome, impacting the storage of genetic information and also its retrieval. While the molecular origin of stacking driving force has been attributed to many different sources including dispersion, electrostatics, and solvent hydrogen bonding, here we show via a systematic decomposition of the stacking free energy using large-scale computer simulations that the dominant driving force stabilizing base stacking is nonhydrophobic solvent entropy. Counteracting this is the conformational entropic penalty on the sugar-phosphate backbone against stacking, while solvent hydrogen-bonding, charge-charge interactions, and dispersive forces produce only secondary perturbations. Solvent entropic forces and DNA backbone conformational strains therefore work against each other, leading to a very mild composite stacking free energy in agreement with experiments.

Mutagenic translesion DNA polymerase V (UmuD′2C) is induced as part of the DNA damage-induced SOS response in Escherichia coli, and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD′2C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD′2C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V Mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which on → off switching is “rapid” at 37°C (~ 1 to 1.5 h), “slow” at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecA E38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro, in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo., Author summary Escherichia coli upregulates more than 40 genes as part of the DNA damage-induced SOS regulon, many of which are involved in DNA repair and cell division. However, three DNA polymerases, pols V, II, and IV, are also induced to rescue replication forks blocked at persisting template lesions. Pol V (UmuD′2C), encoded by the UV mutagenesis genes (umuDC), is primarily responsible for the increase in UV-induced chromosomal mutagenesis. However, pol V is catalytically inert. Interaction with a RecA nucleoprotein filament (RecA*) and ATP is required to convert pol V to an activated “mutasome” complex, pol V Mut = UmuD′2C-RecA-ATP. Here, we show that pol V Mut deactivates dynamically during DNA synthesis, and statically in the absence of synthesis. Activated and deactivated states are governed by a conformational switch that repositions RecA relative to UmuC. Switching rates are more rapid at 37 than at 30°C, and do not require ATP hydrolysis. ATP (ATPγS) binding plays two required regulatory roles: 1) it allows binding of pol V Mut to primer-template DNA; 2) it triggers the RecA-UmuC conformational switch that activates pol V Mut.

While single-stranded (ss) segments of DNAs and RNAs are ubiquitous in biology, details about their structures have only recently begun to emerge. To study ssDNA and RNAs, we have developed a new Monte Carlo (MC) simulation using a free energy model for nucleic acids that has the atomisitic accuracy to capture fine molecular details of the sugar-phosphate backbone. Formulated on the basis of a first-principle calculation of the conformational entropy of the nucleic acid chain, this free energy model correctly reproduced both the long and short length-scale structural properties of ssDNA and RNAs in a rigorous comparison against recent data from fluorescence resonance energy transfer, small-angle X-ray scattering, force spectroscopy and fluorescence correlation transport measurements on sequences up to ∼100 nucleotides long. With this new MC algorithm, we conducted a comprehensive investigation of the entropy landscape of small RNA stem-loop structures. From a simulated ensemble of ∼10(6) equilibrium conformations, the entropy for the initiation of different size RNA hairpin loops was computed and compared against thermodynamic measurements. Starting from seeded hairpin loops, constrained MC simulations were then used to estimate the entropic costs associated with propagation of the stem. The numerical results provide new direct molecular insights into thermodynaimc measurement from macroscopic calorimetry and melting experiments.

A nucleic acid folds according to its free energy, but persistent residual conformational fluctuations remain along its sugar-phosphate backbone even after secondary and tertiary structures have been assembled, and these residual conformational entropies provide a rigorous lower bound for the folding free energy. We extend a recently reported algorithm to calculate the residual backbone entropy along a RNA or DNA given configuration of its bases and apply it to the crystallographic structures of the 23S ribosomal subunit and DNAs in the nucleosome core particle. In the 23S rRNAs, higher entropic strains are concentrated in helices and certain tertiary interaction platforms while residues with high surface accessibility and those not involved in base pairing generally have lower strains. Upon folding, residual backbone entropy in the 23S subunit accounts for an average free energy penalty of +0.47 (kcal/mol)/nt (nt = nucleotide) at 310 K. In nucleosomal DNAs, backbone entropies show periodic oscillations with sequence position correlating with the superhelical twist and shifts in the base-pair-step geometries, and nucleosome positioning on the bound DNA exerts strong influence over where entropic strains are located. In contrast to rRNAs, residual backbone entropies account for a free energy penalty of only +0.09 (kcal/mol)/nt in duplex relative to single-stranded DNAs.

While major contributors to the free energy of RNA tertiary structures such as basepairing, base-stacking, and charge and counterion interactions have been studied extensively, little is known about the intrinsic free energy of the backbone. To assess the magnitude of the entropic strains along the phosphate backbone and their impact on the folding free energy, we have formulated a mathematical treatment for computing the volume of the main-chain torsion-angle conformation space between every pair of nucleobases along any sequence to compute the corresponding backbone entropy. To validate this method, we have compared the computed conformational entropies against a statistical free energy analysis of structures in the crystallographic database from several-thousand backbone conformations between nearest-neighbor nucleobases as well as against extensive computer simulations. Using this calculation, we analyzed the backbone entropy of several ribozymes and riboswitches and found that their entropic strains are highly localized along their sequences. The total entropic penalty due to steric congestions in the backbone for the native fold can be as high as 2.4 cal/K/mol per nucleotide for these medium and large RNAs, producing a contribution to the overall free energy of up to 0.72 kcal/mol per nucleotide. For these RNAs, we found that low-entropy high-strain residues are predominantly located at loops with tight turns and at tertiary interaction platforms with unusual structural motifs.

We formulated a master equation-based mathematical model to analyze random scanning and catalysis for enzymes that act on single-stranded DNA (ssDNA) substrates. Catalytic efficiencies and intrinsic scanning distances are deduced from the distribution of positions and gap lengths between a series of catalytic events occurring over time, which are detected as point mutations in a lacZα-based reporter sequence containing enzyme target motifs. Mathematical analysis of the model shows how scanning motions become separable from the catalysis when the proper statistical properties of the mutation pattern are used to interpret the readouts. Two-point correlations between all catalytic events determine intrinsic scanning distances, whereas gap statistics between mutations determine their catalytic efficiencies. Applying this model to activation-induced deoxycytidine deaminase (AID), which catalyzes C→U deaminations processively on ssDNA, we have established that deaminations of AGC hot motifs occur at a low rate, ∼0.03 s−1, and low efficiency, ∼3%. AID performs random bidirectional movements for an average distance of 6.2 motifs, at a rate of about 15 nucleotides per second, and “dwells” at a motif site for 2.7 s while bound >4 min to the same DNA molecule. These results provide new and important insights on how AID may be optimized for generating mutational diversity in Ig genes, and we discuss how the properties of AID acting freely on a “naked” ssDNA relate to the constrained action of AID during transcription-dependent somatic hypermutation and class-switch recombination.

How to properly account for polyvalent counterions in a molecular dynamics simulation of polyelectrolytes such as nucleic acids remains an open question. Not only do counterions such as Mg(2+) screen electrostatic interactions, they also produce attractive intrachain interactions that stabilize secondary and tertiary structures. Here, we show how a simple force field derived from a recently reported implicit counterion model can be integrated into a molecular dynamics simulation for RNAs to realistically reproduce key structural details of both single-stranded and base-paired RNA constructs. This divalent counterion model is computationally efficient. It works with existing atomistic force fields, or coarse-grained models may be tuned to work with it. We provide optimized parameters for a coarse-grained RNA model that takes advantage of this new counterion force field. Using the new model, we illustrate how the structural flexibility of RNA two-way junctions is modified under different salt conditions.

This paper describes a new all-atom Monte Carlo (MC) simulation program for RNAs. The core numerical engine of this program is powered exclusively by inverse kinematic loop closures. The new simula...

In this paper, we describe how the inverse kinematic solution to the loop closure problem may be generalized to reclose a RNA segment of arbitrary length containing any number of nucleotides without disturbing the atomic positions of the rest of the molecule. This generalization is made possible by representing the boundary conditions of the closure in terms of a set of virtual coordinates called RETO, allowing the inverse kinematics to be reduced from the original six-variable/six-constraint problem to a four-variable/four-constraint problem. Based on this generalized closure solution, a new Monte Carlo algorithm has been formulated and implemented in a fully atomistic RNA simulation capable of moving loops of arbitrary lengths using torsion angle updates exclusively. Combined with other conventional Monte Carlo moves, this new algorithm is able to sample large-scale RNA chain conformations much more efficiently. The utility of this new class of Monte Carlo moves in generating large-loop conformational rearrangements is demonstrated in the simulated unfolding of the full-length hammerhead ribozyme with a bound substrate.

We consider the loop-closure problem for nucleic acids and describe an efficient numerical algorithm for closing single-nucleotide loops in nucleic acids. Using six new internal coordinates to represent the nucleotide conformation, which we call the R-representation, the original closure problem with six free torsion angles in each nucleotide can be reduced to one with only four degrees of freedom. Simple numerical techniques have been used to solve the resulting loop-closure equations, and a test of the closure algorithm on a set of RNAs consisting of more than 7000 nucleotides was able to regenerate the native torsion angles in every nucleotide in the test set without exception. We show how the conformational probability density transforms when the original torsion angle representation is mapped onto the new R-representation. We also present statistical evidence showing that the δ and ν2 torsion angles are coupled, and how this coupling affects the conformation probability density in the R-representation. In addition to the backbone, the same loop-closure algorithm can also be applied to close the ribose ring. The algorithm is freely available at http://tyrosine.use.edu/closure. © 2007 Wiley Periodicals, Inc. J Comput Chem 2008

Any nucleic acid structure that contains single-stranded (ss) segments is dominated by entropy, but molecular-level information about these entropic determinants are inaccessible to direct experiments. A new Monte Carlo (MC) simulation has been developed based on a recently reported backbone conformational formula (BCV/-c) for RNAs. BCV/-c analytically enumerates all conformational states on the sugar-phosphate backbone. It has been validated rigorously against experiments, and the new MC algorithm, NUCLEIC, is atomistically accurate, computationally efficient, as well as predictive. We used NUCLEIC to investigate the entropic landscape of a comprehensive set of RNA secondary structures including: hairpins, stem-loops, symmetric and asymmetric bulges, 2-way, 3-way and 4-way junctions, as well as triplexes and quadruplexes. We found strong evidence that the kinetic barriers to the formation of many of these secondary structural elements are determined by the entropic bottlenecks associated with the unpaired ss tracts on each structure, though the thermodynamic free energy factor that stabilizes them come predominantly from base stacking. Based on these entropy data, we computed the rates of formation of various secondary structures and found that the kinetics of different pathways leading to the same product structure often have significantly different rates. These results highlight the importance of a molecular-level understanding of entropy as a defining factor behind RNA structures.

The complete coding sequence of manganese superoxide dismutase (Mn-SOD) of Trichinella pseudospiralis (Tp) was obtained and characterized for the first time by degenerative reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of complementary DNA ends (RACE) reactions. The open reading frame of Tp Mn-SOD contained 663 nucleotides, encoding 220 amino acid residues. This included the conserved histidine and aspartate residues for metal binding, cysteine residues for disulfide bond formation, and arginine residue for directing the superoxide ion to the protein. The presence of mitochondrial transit peptides and maturation cleavage site suggest that the cloned Tp Mn-SOD gene is a mitochondrial enzyme. It is a single copy gene containing three introns. Northern blotting suggested that the expression level of Mn-SOD is lower than Cu/Zn SOD in infective stage larvae. Semi-quantitative RT-PCR demonstrated that a single dominant transcript of Tp Mn-SOD was highly expressed in the infective-stage larvae but not in adult worms. The information provides a better understanding of the highly compartmentalized superoxide dismutases of adenophorean nematodes.

Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer., Activation-induced deoxycytidine deaminase (AID) induces somatic hypermutation and class-switch recombination during transcription of immunoglobulin genes. Here the authors use single-molecule FRET to show that AID translocates together with RNA polymerase and scans within stalled transcription bubbles.

We present an implicit ion model fo the calculation of the electrostatic free energies of RNA conformations in the presence of divalent counterions such as Mg(2+). The model was applied to the native and several non-native structures of the hammerhead ribozyme and the group I intron in Tetrahymena to study the stability of candidate unfolding intermediates. Based on a rigorous statistical mechanical treatment of the counterions that are closely associated with the RNA while handling the rest of the ions in the solution via a mean field theory in the Grand Canonical ensemble, the implicit ion model accurately reproduces the ordering of their free energies, correctly identifying the native fold as the most stable structure out of the other alternatives. For RNA concentrations in the range below 0.1 μM, divalent concentrations of ∼0.5 mM or above, and over a wide range of solvent dielectric constants, the equilibrium number of divalent ions associated with the RNA remains close to what is needed to exactly neutralize the phosphate negative charges, but the stability of compact RNA folds can be reversed when the divalent ion concentration is lower than ∼0.1 mM, causing the number of associated ions to underneutralize the RNA. In addition to calculating counterion-mediated free energies, the model is also able to identify potential high-affinity electronegative ion binding pockets on the RNA. The model can be easily integrated into an all-atom Monte Carlo RNA simulation as an implicit counterion model.

To elucidate the mechanism of inducing translationally controlled tumor protein (TCTP) in stress adaptation of adenophorean nematodes, the complete coding sequence of TCTP of the infective-stage larvae of Trichinella pseudospiralis was characterized. Two cDNA clones with different 3' untranslated region were identified. Tp-TCTP contained an open reading frame of 534 bp encoding 177 residues. The gene with five introns was expressed as histidine-tagged fusion protein having a molecular mass of 17.5 kDa. Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis showed that TCTP RNA was not accumulated when the infective-stage larvae were heat-shocked for 1 h at 45 or 60 degrees C. Using enzyme-linked immunosorbent assay and antiserum against the fusion protein, the expression of TCTP was found to be up-regulated at the translational level. The data suggest that translational regulation of TCTP may play an important role in the early heat-stress adaptation of the trichinellid. Cluster analysis demonstrated that the TCTP sequence of T. pseudospiralis is closely related to that of T. spiralis, but is diverged from the secernentean species.

Copper/zinc (Cu/Zn) superoxide dismutase (SOD) activity was identified for the first time in both crude somatic extracts (CE) and excretory/secretory (E/S) products of Trichinella pseudospiralis. It was the dominant SOD in infective-stage larvae. Native polyacrylamide gel electrophoresis of CE and E/S products yielded a prominent band, which was cyanide-sensitive and was partly inhibited by hydrogen peroxide in SOD assay. Cytosolic Cu/Zn SOD was cloned. The 471-bp full-length cDNA sequence contained an open reading frame of 157 amino acids. The gene contained three introns. Quantitative reverse transcription-polymerase chain reaction indicated that the expression of cytosolic Cu/Zn SOD was substantially higher in infective-stage larvae than in adult worms. Cluster analysis showed that the sequence of the Cu/Zn SOD of T. pseudospiralis, an adenophorean nematode, is related to those of Brugia pahangi, Acanthocheilonema viteae, Onchocerca volvulus, and Haemonchus contortus (all belonging to the sercenentean group).

We describe a novel simulation method that eliminates the slowing-down problem in the Monte Carlo (MC) simulations of imaginary-time path integrals near the continuum limit. This method combines a stochastic blocking procedure with the multigrid method to rapidly accelerate the sampling of paths in a quantum MC simulation, making its dynamics more ergodic. The effectiveness and efficiency of this method are demonstrated for several one-dimensional quantum systems and compared to other standard and accelerated methods.

Functional RNAs can fold into intricate structures using a number of different secondary and tertiary structural motifs. Many factors contribute to the overall free energy of the target fold. This study aims at quantifying the entropic costs coming from the loss of conformational freedom when the sugar-phosphate backbone is subjected to constraints imposed by secondary and tertiary contacts. Motivated by insights from topology theory, we design a diagrammatic scheme to represent different types of RNA structures so that constraints associated with a folded structure may be segregated into mutually independent subsets, enabling the total conformational entropy loss to be easily calculated as a sum of independent terms. We used high-throughput Monte Carlo simulations to simulate large ensembles of single-stranded RNA sequences in solution to validate the assumptions behind our diagrammatic scheme, examining the entropic costs for hairpin initiation and formation of many multiway junctions. Our diagrammatic scheme aids in the factorization of secondary/tertiary constraints into distinct topological classes and facilitates the discovery of interrelationships among multiple constraints on RNA folds. This perspective, which to our knowledge is novel, leads to useful insights into the inner workings of some functional RNA sequences, demonstrating how they might operate by transforming their structures among different topological classes.

This article provides an introduction to the ideas behind the multilevel blocking (MLB) approach to the fermion sign problem in path-integral Monte Carlo simulations, and also gives a detailed discussion of MLB results for quantum dots. MLB can turn the exponential severity of the sign problem into an algebraic one, thereby enabling numerically exact studies of otherwise inaccessible systems. Low-temperature simulation results for up to eight strongly correlated electrons in a parabolic 2D quantum dot are presented., Comment: 10 Pages, includes 4 figures and mprocl.sty

A novel DNA-binding peptide ofMr∼30 kDa was documented for the first time in the excretory–secretory (E–S) products of the infective-stage larvae ofTrichinella pseudospiralis.Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E–S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of theMrof proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E–S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E–S product.

The multilevel blocking algorithm recently proposed as a possible solution to the sign problem in path-integral Monte Carlo simulations has been extended to systems with long-ranged interactions along the Trotter direction. As an application, new results for the real-time quantum dynamics of the spin-boson model are presented., Comment: 7 pages, 1 figure (eps) to be published in Physical Review E

A novel acidic extracellular single-stranded endonuclease was demonstrated for the first time in the excretory–secretory (E–S) products of 2 species of Trichinella. Unlike the double-stranded endonuclease reported earlier, the single-stranded molecule is divalent cation independent and is detected in both T. spiralis and T. pseudospiralis E–S products. It hydrolysed single-stranded DNA and RNA at comparable rates. The single-stranded endonuclease was sensitive to inhibition by Zn2+ and to high concentrations of NaCl. Zymographic analysis indicated that it was encoded by at least 3 peptides of Mr ∼ 50–60 kDa. The rate of hydrolysis of single-stranded targets by the E–S products was substantially higher than that of the double-stranded molecule. Due to the differences in peptide profile, divalent cation dependence, and species-specific expression, the single and double-stranded endonucleases are likely to be encoded by different proteins and may have different functions.

We present a semiclassical method for simulating the dynamics of nonadiabatic transitions in a discrete-state quantum system coupled to a bath of explicit continuous coordinates. This method employs a coherent-state formulation of the path integrals for the discrete system whose dynamics is described by spin operators. This spin coherent-state formulation allows the discrete system to be mapped onto a continuous coordinate. Stationary approximations of the resulting coherent-state path integrals of the system plus bath lead to quasiclassical equations of motion which can be solved numerically by direct integration. This algorithm reduces the problem to a number of simple classical trajectory calculations and does not require calculating any fluctuation determinants.

We present calculations of the anisotropy of the tension of interfaces separating an isotropic phase rich in a flexible polymer from a nematic phase rich in a semiflexible polymer. We find that the interfacial tension is always lower when the director is parallel to the plane of the interface than when it is perpendicular. The ratio of the tension in the perpendicular to parallel case can be quite large, of order 5 or so, depending on the strength of the nematic interactions. We also find that the interfacial width is always lower in the parallel case even though the tension is lower; this is because the orientational density prefers parallel alignment of chains at the interface.

In this letter we propose a hybrid molecular dynamics/Monte Carlo (MD/MC) method to simulate the statistical behavior of tethered and fluid membranes which does not suffer from severe slowing-down problems as in conventional MC and MD methods. A parallel version of the algorithm allows for reliable determination of the scaling behavior of open and closed membranes.

A transferable tight-binding potential has been constructed for heteroatomic systems containing carbon and hydrogen. The electronic degree of freedom is treated explicitly in this potential using a small set of transferable parameters which has been fitted to small hydrocarbons and radicals. Transferability to other higher hydrocarbons was tested by comparison with ab initio calculations and experimental data. The potential can correctly reproduce changes in the electronic configuration as a function of the local bonding geometry around each carbon atom. This type of potential is well suited for computer simulations of covalently bonded systems in both gas-phase and condensed-phase systems.

We propose a method, Temperature Integration, which allows an efficient calculation of free energy differences between two systems of interest, with the same degrees of freedom, which may have rough energy landscapes. The method is based on calculating, for each single system, the difference between the values of ln Z at two temperatures, using a Parallel Tempering procedure. If our two systems of interest have the same phase space volume, they have the same values of ln Z at high- T , and we can obtain the free energy difference between them, using the two single-system calculations described above. If the phase space volume of a system is known, our method can be used to calculate its absolute (versus relative) free energy as well. We apply our method and demonstrate its efficiency on a “toy model” of hard rods on a 1-dimensional ring.

The mechanism of the ultrafast primary charge separation process in bacterial photosynthesis has been examined with a general dissipative three-state tight-binding model. Using real-time path integrals, the transient populations of the three electronic states (BChl[sub 2]*BChlBPh, BChl[sub 2][sup +]BChl[sup [minus]]BPh, BChl[sub 2][sup +]BChlBPh[sup [minus]]) involved in the reaction have been computed by numerically exact quantum Monte Carlo techniques. The simulations show that the dissipative three-state system can reproduce many characteristic features of the initial charge separation in the reaction center for at least two parameter regions. In both regions, the accessory bacteriochlorophyll population remains small throughout the electron transfer and the transfer rate exhibits the experimentally observed inverse temperature dependence. The first region is associated with a low-lying BChl[sub 2][sup +]BChl[sup [minus]] state and weak electronic couplings. In this region, the transient populations are predominantly monoexponential, and the dynamics is consistent with a stepwise (incoherent) mechanism with nonadiabatic electron-transfer rates. The other region is associated with a BChl[sub 2][sup +]BChl[sup [minus]] state lying above the photoexcited special pair (BChl[sub 2]*). 52 refs., 15 figs., 9 tabs.

Employing a ``noninteracting-cluster approximation,'' the dynamics of multistate dissipative tight-binding models has been formulated in terms of a set of generalized retarded master equations. The rates for the various pathways are expressed as power series in the intersite couplings. We apply this to the superexchange mechanism, which is relevant for bacterial photosynthesis and bridged electron transfer systems. This approach provides a general and unified description of both incoherent and coherent transport.

We study a general asymmetric spin‐boson model for electron transfer processes in the nonadiabatic regime. Our path‐integral approach reduces to the conventional Marcus result in the high‐temperature limit, whereas quantum effects lead to distinct deviations from classical behaviors at low temperatures. Adopting a simple but realistic model spectral density for the environmental modes, we show that for low temperatures, the rate becomes sensitive to the details of the spectral density. In particular, due to nuclear tunneling, the rate maximum does not correspond to the usual classical estimate for the activationless case and the rate is asymmetric around the maximum. A centroid factorization using a properly defined nuclear reaction coordinate provides new insight into the mechanism of nonadiabatic electron transfer.

This paper presents results of path‐integral quantum dynamics simulations of electron transfer rates in a simple class of model Hamiltonians for symmetric and asymmetric electron transfer systems. Our study is aimed at testing the practical usefulness of a centroid factorization of the electron transfer rate constant in the deep tunneling regime. To circumvent the sign problem in quantum Monte Carlo simulations of the electron flux, local filtering techniques have been employed. The simulations show that due to dynamical effects, the reactive flux far outside the transition state region also plays an important role in determining the rate. These results suggest that while the centroid formulation of the equilibrium (imaginary‐time) quantum transition state theory (QTST) applied to the electron path is accurate for nonadiabatic electron transfer reactions, dynamical (real‐time) effects can produce significant corrections to the QTST estimate for the rate outside this region. For model parameters characteri...

Rigorous quantum dynamics simulations have been performed to test the path-integral centroid formulation proposed by Voth, Chandler and Miller for quantal rate constants in a simple class of models for electron transfer reactions in the tight-binding limit. Fully dynamical quantum Monte Carlo simulations show that inside and slightly away from the diabatic limit the electron flux is invariant with the electronic centroid density near the dividing surface instead of being sharply peaked there. Despite this, we show analytically that the rate in this regime is largely governed by the centroid density on the dividing surface. But on approaching the adiabatic limit with larger electronic coupling, complex large-amplitude coherent oscillations develop in the quantum flux, suggesting that in addition to the imaginary-time centroid density, the dynamical factors also play an important role in determining the full quantal electron transfer rate.

A new stochastic method for the direct computation of real-time Green's functions is proposed. The inherent sign problem is circumvented by partitioning the path integration info two parts, one of which involves conventional stochastic sampling, and the other explicit or analytical summation. Using this method, the dynamics of the spin-boson model may be computed up to several tunneling periods. The results reveal surprisingly complex relaxation behaviors near the coherent-incoherent boundary at low temperatures

A practical method to tackle the sign problem in real-time path integral simulations is proposed based on the multilevel blocking idea. The formulation is made possible by using a cumulant expansion of the action, which in addition to addressing the sign problem, provides an unbiased estimator for the action from a statistically noisy sample of real-time paths. The cumulant formulation also allows the analytical gradients of the action to be computed with little extra computational effort, and it can easily be implemented in a massively parallel environment.

Path integral Monte Carlo simulations have been carried out to study the rotations of a methane molecule and its heavier isotopomers inside a small cluster of 4He atoms at 0.3 K in order to determine how the renormalization in the methane's rotational constant is related to the quantum statistics and superfluidity of the helium shell. By changing the mass of the hydrogens and systematically varying the moment of inertia of the methane, we were able to study the effects of its rotations on the quantum statistics of the helium atoms and their countereffects on the methane's effective rotational constant. The renormalized rotational constant depends strongly on the intrinsic moment of inertia of the methane. A heavy probe favors strong templating of the helium density as well as a large renormalization in the probe's rotational constant, but a light probe shows almost no effect on the shell density or the effective rotational constant. These results suggest that in order to fully understand the superfluidity of the helium shell, the probe must be treated as an integral part of the system. We rationalize the findings in terms of a rotational smearing effect and suggest that there is no clearly quantifiable relationship between the superfluid fraction of the shell and the renormalized rotational constant of the probe for cases where the probe molecule is either light or has weak anisotropic interactions with the helium atoms.