The Experts below are selected from a list of 1605 Experts worldwide ranked by ideXlab platform
Samuel E Butcher - One of the best experts on this subject based on the ideXlab platform.
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thermodynamics and folding pathway of Tetraloop receptor mediated rna helical packing
Journal of Molecular Biology, 2008Co-Authors: Kirk Vander A Meulen, Jared H Davis, Trenton R Foster, Thomas M Record, Samuel E ButcherAbstract:Little is known about the thermodynamic forces that drive the folding pathways of higher-order RNA structure. In this study, we employ calorimetric [isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC)] and spectroscopic (NMR and UV) methods to characterize the thermodynamics of the GAAA Tetraloop-receptor interaction, utilizing a previously described bivalent construct. ITC studies indicate that the bivalent interaction is enthalpy driven and highly stable, with a binding constant (K(obs)) of 5.5x10(6) M(-1) and enthalpy (DeltaH(obs)(o)) of -33.8 kcal/mol at 45 degrees C in 20 mM KCl and 2 mM MgCl(2). Thus, we derive the DeltaH(obs)(o) for a single Tetraloop-receptor interaction to be -16.9 kcal/mol at these conditions. UV absorbance data indicate that an increase in base stacking quality contributes to the enthalpy of complex formation. These highly favorable thermodynamics are consistent with the known critical role for the Tetraloop-receptor motif in the folding of large RNAs. Additionally, a significant heat capacity change (DeltaC(p,obs)(o)) of -0.24 kcal mol(-1) K(-1) was determined by ITC. DSC and UV-monitored thermal denaturation experiments indicate that the bivalent Tetraloop-receptor construct follows a minimally five-state unfolding pathway and suggest the observed DeltaC(p,obs)(o) for the interaction results from a temperature-dependent unbound receptor RNA structure.
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role of metal ions in the Tetraloop receptor complex as analyzed by nmr
RNA, 2006Co-Authors: Jared H Davis, Trenton R Foster, Marco Tonelli, Samuel E ButcherAbstract:Metal ions are critical for the proper folding of RNA, and the GAAA Tetraloop–receptor is necessary for the optimal folding and function of many RNAs. We have used NMR to investigate the role of metal ions in the structure of the Tetraloop–receptor in solution. The NMR data indicate native tertiary structure is formed under a wide range of ionic conditions. The lack of conformational adaptation in response to very different ionic conditions argues against a structural role for divalent ions. Nuclear Overhauser effects to cobalt hexammine and paramagnetic relaxation enhancement induced by manganese ions were used to determine the NMR structures of the Tetraloop receptor in association with metal ions, providing the first atomic-level view of these interactions in the solution state. Five manganese and two cobalt hexammine ions could be localized to the RNA surface. The locations of the associated metal ions are similar, but not identical to, those of previously determined crystal structures. The sites of association are in general agreement with nonlinear Poisson–Boltzmann calculations of the electrostatic surface, emphasizing the general importance of diffusely associated ions in RNA tertiary structure.
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rna helical packing in solution nmr structure of a 30 kda gaaa Tetraloop receptor complex
Journal of Molecular Biology, 2005Co-Authors: Jared H Davis, Luc Jaeger, Marco Tonelli, Lincoln G Scott, James R Williamson, Samuel E ButcherAbstract:Tertiary interactions are critical for proper RNA folding and ribozyme catalysis. RNA tertiary structure is often condensed through long-range helical packing interactions mediated by loop-receptor motifs. RNA structures displaying helical packing by loop-receptor interactions have been solved by X-ray crystallography, but not by NMR. Here, we report the NMR structure of a 30 kDa GAAA Tetraloop-receptor RNA complex. In order to stabilize the complex, we used a modular design in which the RNA was engineered to form a homodimer, with each subunit containing a GAAA Tetraloop phased one helical turn apart from its cognate 11-nucleotide receptor domain. The structure determination utilized specific isotopic labeling patterns (2H, 13C and 15N) and refinement against residual dipolar couplings. We observe a unique and highly unusual chemical shift pattern for an adenosine platform interaction that reveals a spectroscopic fingerprint for this motif. The structure of the GAAA Tetraloop-receptor interaction is well defined solely from experimental NMR data, shows minor deviations from previously solved crystal structures, and verifies the previously inferred hydrogen bonding patterns within this motif. This work demonstrates the feasibility of using engineered homodimers as modular systems for the determination of RNA tertiary interactions by NMR.
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a novel family of rna Tetraloop structure forms the recognition site for saccharomyces cerevisiae rnase iii
The EMBO Journal, 2001Co-Authors: Pok Kwan Yang, Samuel E Butcher, Sundeep Kang, Guillaume Chanfreau, Juli FeigonAbstract:RNases III are a family of double-stranded RNA (dsRNA) endoribonucleases involved in the processing and decay of a large number of cellular RNAs as well as in RNA interference. The dsRNA substrates of Saccharomyces cerevisiae RNase III (Rnt1p) are capped by Tetraloops with the consensus sequence AGNN, which act as the primary docking site for the RNase. We have solved the solution structures of two RNA hairpins capped by AGNN Tetraloops, AGAA and AGUU, using NMR spectroscopy. Both Tetraloops have the same overall structure, in which the backbone turn occurs on the 3' side of the syn G residue in the loop, with the first A and G in a 5' stack and the last two residues in a 3' stack. A non-bridging phosphate oxygen and the universal G which are essential for Rnt1p binding are strongly exposed. The compared biochemical and structural analysis of various Tetraloop sequences defines a novel family of RNA Tetraloop fold with the consensus (U/A)GNN and implicates this conserved structure as the primary determinant for specific recognition of Rnt1p substrates.
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quantitative analysis of the isolated gaaa Tetraloop receptor interaction in solution a site directed spin labeling study
Biochemistry, 2001Co-Authors: Peter Z Qin, Samuel E Butcher, Juli Feigon, Wayne L HubbellAbstract:The GNRA (N: any nucleotide; R: purine) Tetraloop/receptor interaction is believed to be one of the most frequently occurring tertiary interaction motifs in RNAs, but an isolated Tetraloop/receptor complex has not been identified in solution. In the present work, site-directed spin labeling is applied to detect Tetraloop/receptor complex formation and estimate the free energy of interaction. For this purpose, the GAAA Tetraloop/receptor interaction was chosen as a model system. A method was developed to place nitroxide labels at specific backbone locations in an RNA hairpin containing the GAAA Tetraloop. Formation of the Tetraloop/receptor complex was monitored through changes in the rotational correlation time of the Tetraloop and the attached nitroxide. Results show that a hairpin containing the GAAA Tetraloop forms a complex with an RNA containing the 11-nucleotide GAAA Tetraloop receptor motif with an apparent Kd that is strongly dependent on Mg2+. At 125 mM MgCl2, Kd = 0.40 +/- 0.05 mM. The corresponding standard free energy of complex formation is -4.6 kcal/mol, representing the energetics of the Tetraloop/receptor interaction in the absence of other tertiary constraints. The experimental strategy presented here should have broad utility in quantifying weak interactions that would otherwise be undetectable, for both nucleic acids and nucleic acid-protein complexes.
Juli Feigon - One of the best experts on this subject based on the ideXlab platform.
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site directed spin labeling studies reveal solution conformational changes in a gaaa Tetraloop receptor upon mg2 dependent docking of a gaaa Tetraloop
Journal of Molecular Biology, 2005Co-Authors: Peter Z Qin, Juli Feigon, Wayne L HubbellAbstract:The Mg2+-dependent GAAA Tetraloop interaction with its 11 nucleotide receptor is one of the most frequently occurring long-range tertiary interactions in RNAs. To explore conformational changes in the receptor during Tetraloop docking, nitroxide spin labels were attached at each of four uridine bases, one at a time, within an RNA molecule containing the receptor sequence. In the presence of Mg2+ and the Tetraloop, the electron paramagnetic resonance (EPR) spectrum of one of the labeled bases reflected a large increase in mobility, indicating unstacking of the base upon Tetraloop docking. This provides direct evidence that base unstacking is an intrinsic feature of the solution Tetraloop–receptor complex formed in the presence of Mg2+. Additional evidence suggests that in solution the bound receptor conformation is similar to that observed in the crystal structure of a group I intron ribozyme domain. In Mg2+ alone, a receptor conformation with an unstacked base was not detectable, suggesting that this conformation is of higher standard state free energy than that of the free receptor. This leads to the conclusion that the extensive RNA–RNA interactions observed in the crystal structure of the Tetraloop–receptor complex provide larger interaction energy than the measured apparent affinity between the Tetraloop and the free receptor. This is compatible with a high specificity of the Tetraloop–receptor interaction.
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structural basis for recognition of the agnn Tetraloop rna fold by the double stranded rna binding domain of rnt1p rnase iii
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Anthony K Henras, Guillaume Chanfreau, Juli FeigonAbstract:Specific recognition of double-stranded RNA (dsRNA) by dsRNA-binding domains (dsRBDs) is involved in a large number of biological and regulatory processes. Although structures of dsRBDs in complex with dsRNA have revealed how they can bind to dsRNA in general, these do not explain how a dsRBD can recognize specific RNAs. Rnt1p, a member of the RNase III family of dsRNA endonucleases, is a key component of the Saccharomyces cerevisiae RNA-processing machinery. The Rnt1p dsRBD has been implicated in targeting this endonuclease to its RNA substrates, by recognizing hairpins closed by AGNN Tetraloops. We report the solution structure of Rnt1p dsRBD complexed to the 5′ terminal hairpin of one of its small nucleolar RNA substrates, the snR47 precursor. The conserved AGNN Tetraloop fold is retained in the protein-RNA complex. The dsRBD contacts the RNA at successive minor, major, and Tetraloop minor grooves on one face of the helix. Surprisingly, neither the universally conserved G nor the highly conserved A are recognized by specific hydrogen bonds to the bases. Rather, the N-terminal helix fits snugly into the minor groove of the RNA Tetraloop and top of the stem, interacting in a non-sequence-specific manner with the sugar-phosphate backbone and the two nonconserved Tetraloop bases. Mutational analysis of residues that contact the Tetraloop region show that they are functionally important for RNA processing in the context of the entire protein in vivo. These results show how a single dsRBD can convey specificity for particular RNA targets, by structure specific recognition of a conserved Tetraloop fold.
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a novel family of rna Tetraloop structure forms the recognition site for saccharomyces cerevisiae rnase iii
The EMBO Journal, 2001Co-Authors: Pok Kwan Yang, Samuel E Butcher, Sundeep Kang, Guillaume Chanfreau, Juli FeigonAbstract:RNases III are a family of double-stranded RNA (dsRNA) endoribonucleases involved in the processing and decay of a large number of cellular RNAs as well as in RNA interference. The dsRNA substrates of Saccharomyces cerevisiae RNase III (Rnt1p) are capped by Tetraloops with the consensus sequence AGNN, which act as the primary docking site for the RNase. We have solved the solution structures of two RNA hairpins capped by AGNN Tetraloops, AGAA and AGUU, using NMR spectroscopy. Both Tetraloops have the same overall structure, in which the backbone turn occurs on the 3' side of the syn G residue in the loop, with the first A and G in a 5' stack and the last two residues in a 3' stack. A non-bridging phosphate oxygen and the universal G which are essential for Rnt1p binding are strongly exposed. The compared biochemical and structural analysis of various Tetraloop sequences defines a novel family of RNA Tetraloop fold with the consensus (U/A)GNN and implicates this conserved structure as the primary determinant for specific recognition of Rnt1p substrates.
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quantitative analysis of the isolated gaaa Tetraloop receptor interaction in solution a site directed spin labeling study
Biochemistry, 2001Co-Authors: Peter Z Qin, Samuel E Butcher, Juli Feigon, Wayne L HubbellAbstract:The GNRA (N: any nucleotide; R: purine) Tetraloop/receptor interaction is believed to be one of the most frequently occurring tertiary interaction motifs in RNAs, but an isolated Tetraloop/receptor complex has not been identified in solution. In the present work, site-directed spin labeling is applied to detect Tetraloop/receptor complex formation and estimate the free energy of interaction. For this purpose, the GAAA Tetraloop/receptor interaction was chosen as a model system. A method was developed to place nitroxide labels at specific backbone locations in an RNA hairpin containing the GAAA Tetraloop. Formation of the Tetraloop/receptor complex was monitored through changes in the rotational correlation time of the Tetraloop and the attached nitroxide. Results show that a hairpin containing the GAAA Tetraloop forms a complex with an RNA containing the 11-nucleotide GAAA Tetraloop receptor motif with an apparent Kd that is strongly dependent on Mg2+. At 125 mM MgCl2, Kd = 0.40 +/- 0.05 mM. The corresponding standard free energy of complex formation is -4.6 kcal/mol, representing the energetics of the Tetraloop/receptor interaction in the absence of other tertiary constraints. The experimental strategy presented here should have broad utility in quantifying weak interactions that would otherwise be undetectable, for both nucleic acids and nucleic acid-protein complexes.
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solution structure of a gaaa Tetraloop receptor rna
The EMBO Journal, 1997Co-Authors: Samuel E Butcher, Thorsten Dieckmann, Juli FeigonAbstract:The GAAA Tetraloop receptor is an 11‐nucleotide RNA sequence that participates in the tertiary folding of a variety of large catalytic RNAs by providing a specific binding site for GAAA Tetraloops. Here we report the solution structure of the isolated Tetraloop receptor as solved by multidimensional, heteronuclear magnetic resonance spectroscopy. The internal loop of the Tetraloop receptor has three adenosines stacked in a cross‐strand or zipper‐like fashion. This arrangement produces a high degree of base stacking within the asymmetric internal loop without extrahelical bases or kinking the helix. Additional interactions within the internal loop include a U·U mismatch pair and a G·U wobble pair. A comparison with the crystal structure of the receptor RNA bound to its Tetraloop shows that a conformational change has to occur upon Tetraloop binding, which is in good agreement with previous biochemical data. A model for an alternative binding site within the receptor is proposed based on the NMR structure, phylogenetic data and previous crystallographic structures of Tetraloop interactions.
Angel E Garcia - One of the best experts on this subject based on the ideXlab platform.
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concentration dependent and configuration dependent interactions of monovalent ions with an rna Tetraloop
Journal of Chemical Physics, 2018Co-Authors: Jacob C. Miner, Angel E GarciaAbstract:Monovalent salt solutions have strongly coupled interactions with biopolymers, from large polyelectrolytes to small RNA oligomers. High salt concentrations have been known to induce transitions in the structure of RNA, producing non-canonical configurations and even driving RNA to precipitate out of solution. Using all-atom molecular dynamics simulations, we model a monovalent salt species (KCL) at high concentrations (0.1-3m) and calculate the equilibrium distributions of water and ions around a small Tetraloop-forming RNA oligomer in a variety of structural arrangements: folded A-RNA (canonical) and Z-RNA (non-canonical) Tetraloops and unfolded configurations. From these data, we calculate the ion preferential binding coefficients and Donnan coefficients for the RNA oligomer as a function of concentration and structure. We find that cation accumulation is highest around non-canonical Z-RNA configurations at concentrations below 0.5m, while unfolded configurations accumulate the most co-ions in all concentrations. By contrast, canonical A-RNA structures consistently show the lowest accumulations for all ion species. Water distributions vary markedly with RNA configuration but show little dependency on KCL concentration. Based on Donnan coefficient calculations, the net charge of the solution at the surface of the RNA decreases linearly as a function of salt concentration and becomes net-neutral near 2.5-3m KCL for folded configurations, while unfolded configurations still show a positive solution charge. Our findings show that all-atom molecular dynamics can describe the equilibrium distributions of monovalent salt in the presence of small RNA oligomers at KCL concentrations where ion correlation effects become important. Furthermore, these results provide valuable insights into the distributions of water and ions near the RNA oligomer surface as a function of structural configuration.
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Free-energy landscape of a hyperstable RNA Tetraloop.
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Jacob C. Miner, Alan A. Chen, Angel E GarciaAbstract:We report the characterization of the energy landscape and the folding/unfolding thermodynamics of a hyperstable RNA Tetraloop obtained through high-performance molecular dynamics simulations at microsecond timescales. Sampling of the configurational landscape is conducted using temperature replica exchange molecular dynamics over three isochores at high, ambient, and negative pressures to determine the thermodynamic stability and the free-energy landscape of the Tetraloop. The simulations reveal reversible folding/unfolding transitions of the Tetraloop into the canonical A-RNA conformation and the presence of two alternative configurations, including a left-handed Z-RNA conformation and a compact purine Triplet. Increasing hydrostatic pressure shows a stabilizing effect on the A-RNA conformation and a destabilization of the left-handed Z-RNA. Our results provide a comprehensive description of the folded free-energy landscape of a hyperstable RNA Tetraloop and highlight the significant advances of all-atom molecular dynamics in describing the unbiased folding of a simple RNA secondary structure motif.
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molecular simulations studies of rna Tetraloop hyperstability the effect of stem length on folding dynamics
Biophysical Journal, 2015Co-Authors: Jacob C. Miner, Alan A. Chen, Angel E GarciaAbstract:RNA hairpins, formed by looping oligonucleotides, are among the most common RNA secondary structures and involve conserved interactions that drive hierarchical folding and stabilize folded states. Recent modifications to all-atom molecular dynamics force fields have provided the means to quantitatively assess RNA hairpin folding and stability for comparison with experiment. This study focuses on using these techniques to analyze two hyperstable RNA hairpins, each containing a hyperstable Tetraloop sequence (rGCAA) and a G-C stem of two or four basepairs. The systems were simulated for a total of 300 microseconds using replica exchange molecular dynamics, and the resulting structure ensembles were analyzed for common folded states, intra-loop hydrogen bonds, and stem base-pairing retention. We find that the longer stem produces kinetic traps with non-native loop conformations, while offering credence to supposed alternative Tetraloop folds from experiment.Shorter stems produce more variable folding behaviors and demonstrate reversible folding/unfolding actions. These results offer an unbiased, thermodynamic characterization of RNA Tetraloops, highlight the presence of kinetic traps, and provide critical information for kinetic folding studies of Tetraloops as well as additional folding studies of larger RNA molecules.
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Molecular Simulations of the Folding Kinetics of an RNA Tetraloop
Biophysical Journal, 2009Co-Authors: Dong Guo, Angel E GarciaAbstract:We study the folding kinetics of the r(GCUUCGGC) RNA Tetraloop by combining molecular dynamics and replica exchange molecular dynamics simulations. An 11 microsecond REMD simulation with 52 replicas provides sufficient data to define the dominant conformations of the Tetraloop. We use a clustering analysis method to identify these dominant states; both RMSD and Hamming Distance are used in the clustering. During the REMD simulation all replicas show multiple folding/unfolding events. The REMD trajectories along constant T segments of the simulations are combined with constant T MD simulations to obtain information about transition among states leading from the unfolded to the folded state. A model for the folding kinetics will be described.This work is funded by the National Science Foundation MCB-0543769.
David J Nesbitt - One of the best experts on this subject based on the ideXlab platform.
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an rna folding motif gnra Tetraloop receptor interactions
Quarterly Reviews of Biophysics, 2013Co-Authors: Julie L Fiore, David J NesbittAbstract:Nearly two decades after Westhof and Michel first proposed that RNA Tetraloops may interact with distal helices, Tetraloop–receptor interactions have been recognized as ubiquitous elements of RNA tertiary structure. The unique architecture of GNRA Tetraloops (N=any nucleotide, R=purine) enables interaction with a variety of receptors, e.g., helical minor grooves and asymmetric internal loops. The most common example of the latter is the GAAA Tetraloop–11 nt Tetraloop receptor motif. Biophysical characterization of this motif provided evidence for the modularity of RNA structure, with applications spanning improved crystallization methods to RNA tectonics. In this review, we identify and compare types of GNRA Tetraloop–receptor interactions. Then we explore the abundance of structural, kinetic, and thermodynamic information on the frequently occurring and most widely studied GAAA Tetraloop–11 nt receptor motif. Studies of this interaction have revealed powerful paradigms for structural assembly of RNA, as well as providing new insights into the roles of cations, transition states and protein chaperones in RNA folding pathways. However, further research will clearly be necessary to characterize other Tetraloop–receptor and long-range tertiary binding interactions in detail – an important milestone in the quantitative prediction of free energy landscapes for RNA folding.
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enthalpy driven rna folding single molecule thermodynamics of Tetraloop receptor tertiary interaction
Biochemistry, 2009Co-Authors: Julie L Fiore, Benedikt Kraemer, Felix Koberling, Rainer Edmann, David J NesbittAbstract:RNA folding thermodynamics are crucial for structure prediction, which requires characterization of both enthalpic and entropic contributions of tertiary motifs to conformational stability. We explore the temperature dependence of RNA folding due to the ubiquitous GAAA Tetraloop-receptor docking interaction, exploiting immobilized and freely diffusing single-molecule fluorescence resonance energy transfer (smFRET) methods. The equilibrium constant for intramolecular docking is obtained as a function of temperature (T = 21-47 degrees C), from which a van't Hoff analysis yields the enthalpy (DeltaH degrees) and entropy (DeltaS degrees) of docking. Tetraloop-receptor docking is significantly exothermic and entropically unfavorable in 1 mM MgCl(2) and 100 mM NaCl, with excellent agreement between immobilized (DeltaH degrees = -17.4 +/- 1.6 kcal/mol, and DeltaS degrees = -56.2 +/- 5.4 cal mol(-1) K(-1)) and freely diffusing (DeltaH degrees = -17.2 +/- 1.6 kcal/mol, and DeltaS degrees = -55.9 +/- 5.2 cal mol(-1) K(-1)) species. Kinetic heterogeneity in the Tetraloop-receptor construct is unaffected over the temperature range investigated, indicating a large energy barrier for interconversion between the actively docking and nondocking subpopulations. Formation of the Tetraloop-receptor interaction can account for approximately 60% of the DeltaH degrees and DeltaS degrees of P4-P6 domain folding in the Tetrahymena ribozyme, suggesting that it may act as a thermodynamic clamp for the domain. Comparison of the isolated Tetraloop-receptor and other tertiary folding thermodynamics supports a theme that enthalpy- versus entropy-driven folding is determined by the number of hydrogen bonding and base stacking interactions.
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metal ion dependence thermodynamics and kinetics for intramolecular docking of a gaaa Tetraloop and receptor connected by a flexible linker
Biochemistry, 2006Co-Authors: Christopher D Downey, Julie L Fiore, David J Nesbitt, Colby D Stoddard, Jose H Hodak, Arthur PardiAbstract:The GAAA Tetraloop-receptor is a commonly occurring tertiary interaction motif in RNA. This motif usually occurs in combination with other tertiary interactions in complex RNA structures. Thus, it is difficult to measure directly the contribution that a single GAAA Tetraloop-receptor interaction makes to the folding properties of an RNA. To investigate the kinetics and thermodynamics for the isolated interaction, a GAAA Tetraloop domain and receptor domain were connected by a single-stranded A7 linker. Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA Tetraloop and receptor. Docking was induced using a variety of metal ions, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking ([Co(NH3)63+] ≪ [Ca2+], [Mg2+], [Mn2+] ≪ [Na+], [K+]). Analysis of metal ion cooperativity yielded Hill coefficients of ≈ 2 for Na+- or K+-dependent docking versus ≈ 1 for the divalent ions and Co(NH3)63+. Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA Tetraloop-receptor docking. RNA constructs with U7 and A14 single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking. These studies showed that the single-stranded region functions primarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker was also investigated. The influence of flexible versus rigid linkers on GAAA Tetraloop-receptor docking is discussed.
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docking kinetics and equilibrium of a gaaa Tetraloop receptor motif probed by single molecule fret
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Jose H Hodak, Julie L Fiore, David J Nesbitt, Christopher D Downey, Arthur PardiAbstract:Docking kinetics and equilibrium of fluorescently labeled RNA molecules are studied with single-molecule FRET methods. Time-resolved FRET is used to monitor docking/undocking transitions for RNAs containing a single GAAA Tetraloop-receptor tertiary interaction connected by a flexible single-stranded linker. The rate constants for docking and undocking are measured as a function of Mg2+, revealing a complex dependence on metal ion concentration. Despite the simplicity of this model system, conformational heterogeneity similar to that noted in more complex RNA systems is observed; relatively rapid docking/undocking transitions are detected for approximately two-thirds of the RNA molecules, with significant subpopulations exhibiting few or no transitions on the 10- to 30-s time scale for photobleaching. The rate constants are determined from analysis of probability densities, which allows a much wider range of time scales to be analyzed than standard histogram procedures. The data for the GAAA Tetraloop receptor are compared with kinetic and equilibrium data for other RNA tertiary interactions.
Sandro Bottaro - One of the best experts on this subject based on the ideXlab platform.
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integrating nmr and simulations reveals motions in the uucg Tetraloop
Nucleic Acids Research, 2020Co-Authors: Sandro Bottaro, Parker J Nichols, Beat Vogeli, Michele Parrinello, Kresten LindorfflarsenAbstract:We provide an atomic-level description of the structure and dynamics of the UUCG RNA stem-loop by combining molecular dynamics simulations with experimental data. The integration of simulations with exact nuclear Overhauser enhancements data allowed us to characterize two distinct states of this molecule. The most stable conformation corresponds to the consensus three-dimensional structure. The second state is characterized by the absence of the peculiar non-Watson-Crick interactions in the loop region. By using machine learning techniques we identify a set of experimental measurements that are most sensitive to the presence of non-native states. We find that although our MD ensemble, as well as the consensus UUCG Tetraloop structures, are in good agreement with experiments, there are remaining discrepancies. Together, our results show that (i) the MD simulation overstabilize a non-native loop conformation, (ii) eNOE data support its presence with a population of ≈10% and (iii) the structural interpretation of experimental data for dynamic RNAs is highly complex, even for a simple model system such as the UUCG Tetraloop.
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unexpected dynamics in the uucg rna Tetraloop
bioRxiv, 2019Co-Authors: Sandro Bottaro, Parker J Nichols, Beat Vogeli, Michele Parrinello, Kresten LindorfflarsenAbstract:Abstract Many RNA molecules are dynamic, but characterizing their motions by experiments is difficult, often requiring application of complex NMR experiments. Computational methods such as molecular dynamics simulations, on the other hand, still suffer from difficulties in sampling and remaining force field errors. Here, we provide an atomic-level description of structure and dynamics of the 14-mer UUCG RNA stem-loop by combining molecular dynamics simulations with exact nuclear Overhauser enhancement data. The integration of experiments and simulation via a Bayesian/Maximum entropy approach enables us to discover and characterize a new state of this molecule, which we show samples two distinct states. The most stable conformation corresponds to the native, consensus three-dimensional structure. The second, minor state has a population of 11%, and is characterized by the absence of the peculiar non-Watson-Crick base pair between U and G in the loop region. By using machine learning techniques, we identify key contacts in the NOESY spectrum that are compatible with the presence of the low-populated state. Together, our results demonstrate the validity of our integrative approach to determine the structure and thermodynamics of conformational changes in RNA molecules.
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mapping the universe of rna Tetraloop folds
Biophysical Journal, 2017Co-Authors: Sandro Bottaro, Kresten LindorfflarsenAbstract:We report a map of RNA Tetraloop conformations constructed by calculating pairwise distances among all experimentally determined four-nucleotide hairpin loops. Tetraloops with similar structures are clustered together and, as expected, the two largest clusters are the canonical GNRA and UNCG folds. We identify clusters corresponding to known Tetraloop folds such as GGUG, RNYA, AGNN, and CUUG. These clusters are represented in a simple two-dimensional projection that recapitulates the relationship among the different folds. The cluster analysis also identifies 20 novel Tetraloop folds that are peculiar to specific positions in ribosomal RNAs and that are stabilized by tertiary interactions. In our RNA Tetraloop database we find a significant number of non-GNRA and non-UNCG sequences adopting the canonical GNRA and UNCG folds. Conversely, we find a significant number of GNRA and UNCG sequences adopting non-GNRA and non-UNCG folds. Our analysis demonstrates that there is not a simple one-to-one, but rather a many-to-many mapping between Tetraloop sequence and Tetraloop fold.
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computer folding of rna Tetraloops identification of key force field deficiencies
Journal of Chemical Theory and Computation, 2016Co-Authors: Petra Kuhrova, Sandro Bottaro, Michal Otyepka, Robert B. Best, Giovanni Bussi, Jiři Sponer, Pavel BanasAbstract:The computer-aided folding of biomolecules, particularly RNAs, is one of the most difficult challenges in computational structural biology. RNA Tetraloops are fundamental RNA motifs playing key roles in RNA folding and RNA–RNA and RNA–protein interactions. Although state-of-the-art Molecular Dynamics (MD) force fields correctly describe the native state of these Tetraloops as a stable free-energy basin on the microsecond time scale, enhanced sampling techniques reveal that the native state is not the global free energy minimum, suggesting yet unidentified significant imbalances in the force fields. Here, we tested our ability to fold the RNA Tetraloops in various force fields and simulation settings. We employed three different enhanced sampling techniques, namely, temperature replica exchange MD (T-REMD), replica exchange with solute tempering (REST2), and well-tempered metadynamics (WT-MetaD). We aimed to separate problems caused by limited sampling from those due to force-field inaccuracies. We found t...
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Computer Folding of RNA Tetraloops: Identification of Key Force Field Deficiencies
2016Co-Authors: Petra Kührová, Sandro Bottaro, Jiří Šponer, Michal Otyepka, Robert B. Best, Giovanni Bussi, Pavel BanášAbstract:The computer-aided folding of biomolecules, particularly RNAs, is one of the most difficult challenges in computational structural biology. RNA Tetraloops are fundamental RNA motifs playing key roles in RNA folding and RNA–RNA and RNA–protein interactions. Although state-of-the-art Molecular Dynamics (MD) force fields correctly describe the native state of these Tetraloops as a stable free-energy basin on the microsecond time scale, enhanced sampling techniques reveal that the native state is not the global free energy minimum, suggesting yet unidentified significant imbalances in the force fields. Here, we tested our ability to fold the RNA Tetraloops in various force fields and simulation settings. We employed three different enhanced sampling techniques, namely, temperature replica exchange MD (T-REMD), replica exchange with solute tempering (REST2), and well-tempered metadynamics (WT-MetaD). We aimed to separate problems caused by limited sampling from those due to force-field inaccuracies. We found that none of the contemporary force fields is able to correctly describe folding of the 5′-GAGA-3′ Tetraloop over a range of simulation conditions. We thus aimed to identify which terms of the force field are responsible for this poor description of TL folding. We showed that at least two different imbalances contribute to this behavior, namely, overstabilization of base–phosphate and/or sugar–phosphate interactions and underestimated stability of the hydrogen bonding interaction in base pairing. The first artifact stabilizes the unfolded ensemble, while the second one destabilizes the folded state. The former problem might be partially alleviated by reparametrization of the van der Waals parameters of the phosphate oxygens suggested by Case et al., while in order to overcome the latter effect we suggest local potentials to better capture hydrogen bonding interactions
