The Experts below are selected from a list of 312 Experts worldwide ranked by ideXlab platform
Richard A. Collins - One of the best experts on this subject based on the ideXlab platform.
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additional roles of a peripheral loop loop interaction in the neurospora VS Ribozyme
Nucleic Acids Research, 2011Co-Authors: Diane M. Deabreu, Joan E. Olive, Richard A. CollinsAbstract:Many RNAs contain tertiary interactions that contribute to folding the RNA into its functional 3D structure. In the VS Ribozyme, a tertiary loop-loop kissing interaction involving stem-loops I and V is also required to rearrange the secondary structure of stem-loop I such that nucleotides at the base of stem I, which contains the cleavage-ligation site, can adopt the conformation required for activity. In the current work, we have used mutants that constitutively adopt the catalytically permissive conformation to search for additional roles of the kissing interaction in vitro. Using mutations that disrupt or restore the kissing interaction, we find that the kissing interaction contributes ~1000-fold enhancement to the rates of cleavage and ligation. Large Mg(2+)-dependent effects on equilibrium were also observed: in the presence of the kissing interaction cleavage is favored >10-fold at micromolar concentrations of Mg(2+); whereas ligation is favored >10-fold at millimolar concentrations of Mg(2+). In the absence of the kissing interaction cleavage exceeds ligation at all concentrations of Mg(2+). These data provide evidence that the kissing interaction strongly affects the observed cleavage and ligation rate constants and the cleavage-ligation equilibrium of the Ribozyme.
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Additional roles of a peripheral loop–loop interaction in the Neurospora VS Ribozyme
Nucleic acids research, 2011Co-Authors: Diane M. Deabreu, Joan E. Olive, Richard A. CollinsAbstract:Many RNAs contain tertiary interactions that contribute to folding the RNA into its functional 3D structure. In the VS Ribozyme, a tertiary loop-loop kissing interaction involving stem-loops I and V is also required to rearrange the secondary structure of stem-loop I such that nucleotides at the base of stem I, which contains the cleavage-ligation site, can adopt the conformation required for activity. In the current work, we have used mutants that constitutively adopt the catalytically permissive conformation to search for additional roles of the kissing interaction in vitro. Using mutations that disrupt or restore the kissing interaction, we find that the kissing interaction contributes ~1000-fold enhancement to the rates of cleavage and ligation. Large Mg(2+)-dependent effects on equilibrium were also observed: in the presence of the kissing interaction cleavage is favored >10-fold at micromolar concentrations of Mg(2+); whereas ligation is favored >10-fold at millimolar concentrations of Mg(2+). In the absence of the kissing interaction cleavage exceeds ligation at all concentrations of Mg(2+). These data provide evidence that the kissing interaction strongly affects the observed cleavage and ligation rate constants and the cleavage-ligation equilibrium of the Ribozyme.
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Use of Ribozyme cleavage kinetics to measure salt-induced changes in solution pH.
Analytical biochemistry, 2011Co-Authors: M. Duane Smith, Richard A. CollinsAbstract:Abstract Several Ribozymes are active in molar concentrations of monovalent salts, and pH rate curves under such conditions are consistent with mechanisms involving general acid–base catalysis. Interpreting the apparent pKa values requires an accurate estimation of solution pH, which can be difficult to obtain using a typical glass pH electrode in the presence of high salt concentrations. In the current work we have used the VS Ribozyme as a “biological pH meter” to evaluate the effects of molar concentrations of monovalent salts on changes in solution pH. We find that almost all of the measured change in pH observed in high concentrations of LiCl is due to a real change in solution pH. In contrast, high concentrations of NaCl cause errors in pH measurement in addition to changes in actual pH. Different buffer systems affect both the direction and the magnitude of pH changes: we observed changes in measured pH of up to 1 pH unit, which require proper interpretation to avoid adverse effects on the conclusions drawn from pH rate and other experiments that utilize very high salt concentrations.
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The ionic environment determines Ribozyme cleavage rate by modulation of nucleobase pK a
RNA (New York N.Y.), 2008Co-Authors: M. Duane Smith, Joan E. Olive, Reza Mehdizadeh, Richard A. CollinsAbstract:Several small Ribozymes employ general acid–base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK a values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS Ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK a values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid–base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid–base catalysis indicate that k 1, the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in Ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS Ribozyme can be explained solely by the effect of the ions on nucleobase pK a. The inference that k 1 is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK a is that of the general acid and the higher pK a is that of the general base.
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Single VS Ribozyme molecules reveal dynamic and hierarchical folding toward catalysis.
Journal of molecular biology, 2008Co-Authors: Miguel J. B. Pereira, Evgenia N. Nikolova, Shawna L. Hiley, Dominic C. J. Jaikaran, Richard A. Collins, Nils G. WalterAbstract:Non-coding RNAs of complex tertiary structure are involved in numerous aspects of the replication and processing of genetic information in many organisms; however, an understanding of the complex relationship between their structural dynamics and function is only slowly emerging. The Neurospora Varkud Satellite (VS) Ribozyme provides a model system to address this relationship. First, it adopts a tertiary structure assembled from common elements, a kissing loop and two three-way junctions. Second, catalytic activity of the Ribozyme is essential for replication of VS RNA in vivo and can be readily assayed in vitro. Here we exploit single molecule FRET to show that the VS Ribozyme exhibits previously unobserved dynamic and heterogeneous hierarchical folding into an active structure. Readily reversible kissing loop formation combined with slow cleavage of the upstream substrate helix suggests a model whereby the structural dynamics of the VS Ribozyme favor cleavage of the substrate downstream of the Ribozyme core instead. This preference is expected to facilitate processing of the multimeric RNA replication intermediate into circular VS RNA, which is the predominant form observed in vivo.
Pascale Legault - One of the best experts on this subject based on the ideXlab platform.
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A multi-axial RNA joint with a large range of motion promotes sampling of an active Ribozyme conformation
Nucleic acids research, 2019Co-Authors: Nicolas Girard, Julie Lacroix-labonté, Pierre Dagenais, Pascale LegaultAbstract:Investigating the dynamics of structural elements in functional RNAs is important to better understand their mechanism and for engineering RNAs with novel functions. Previously, we performed rational engineering studies with the Varkud satellite (VS) Ribozyme and switched its specificity toward non-natural hairpin substrates through modification of a critical kissing-loop interaction (KLI). We identified functional VS Ribozyme variants with surrogate KLIs (ribosomal RNA L88/L22 and human immunodeficiency virus-1 TAR/TAR*), but they displayed ∼100-fold lower cleavage activity. Here, we characterized the dynamics of KLIs to correlate dynamic properties with function and improve the activity of designer Ribozymes. Using temperature replica exchange molecular dynamics, we determined that the natural KLI in the VS Ribozyme supports conformational sampling of its closed and active state, whereas the surrogate KLIs display more restricted motions. Based on in vitro selection, the cleavage activity of a VS Ribozyme variant with the TAR/TAR* KLI could be markedly improved by partly destabilizing the KLI but increasing conformation sampling. We formulated a mechanistic model for substrate binding in which the KLI dynamics contribute to formation of the active site. Our model supports the modular nature of RNA in which subdomain structure and dynamics contribute to define the thermodynamics and kinetics relevant to RNA function.
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Insights into RNA structure and dynamics from recent NMR and X-ray studies of the Neurospora Varkud satellite Ribozyme.
Wiley interdisciplinary reviews. RNA, 2017Co-Authors: Pierre Dagenais, Nicolas Girard, Eric Bonneau, Pascale LegaultAbstract:Despite the large number of noncoding RNAs and their importance in several biological processes, our understanding of RNA structure and dynamics at atomic resolution is still very limited. Like many other RNAs, the Neurospora Varkud satellite (VS) Ribozyme performs its functions through dynamic exchange of multiple conformational states. More specifically, the VS Ribozyme recognizes and cleaves its stem-loop substrate via a mechanism that involves several structural transitions within its stem-loop substrate. The recent publications of high-resolution structures of the VS Ribozyme, obtained by NMR spectroscopy and X-ray crystallography, offer an opportunity to integrate the data and closely examine the structural and dynamic properties of this model RNA system. Notably, these investigations provide a valuable example of the divide-and-conquer strategy for structural and dynamic characterization of a large RNA, based on NMR structures of several individual subdomains. The success of this divide-and-conquer approach reflects the modularity of RNA architecture and the great care taken in identifying the independently-folding modules. Together with previous biochemical and biophysical characterizations, the recent NMR and X-ray studies provide a coherent picture into how the VS Ribozyme recognizes its stem-loop substrate. Such in-depth characterization of this RNA enzyme will serve as a model for future structural and engineering studies of dynamic RNAs and may be particularly useful in planning divide-and-conquer investigations. WIREs RNA 2017, 8:e1421. doi: 10.1002/wrna.1421 For further resources related to this article, please visit the WIREs website.
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NMR Localization of Divalent Cations at the Active Site of the Neurospora VS Ribozyme Provides Insights into RNA−Metal-Ion Interactions
2016Co-Authors: Eric Bonneau, Pascale LegaultAbstract:ABSTRACT: Metal cations represent key elements of RNA structure and function. In the Neurospora VS Ribozyme, metal cations play diverse roles; they are important for substrate recognition, formation of the active site, and shifting the pKa’s of two key nucleobases that contribute to the general acid− base mechanism. Recently, we determined the NMR structure of the A730 loop of the VS Ribozyme active site (SLVI) that contributes the general acid (A756) in the enzymatic mechanism of the cleavage reaction. Our studies showed that magnesium (Mg2+) ions are essential to stabilize the formation of the S-turn motif within the A730 loop that exposes the A756 nucleobase for catalysis. In this article, we extend these NMR investigations by precisely mapping the Mg2+-ion binding sites using manganese-induced paramagnetic relaxation enhancement and cadmium-induced chemical-shift perturbation of phosphorothioate RNAs. These experiments identify five Mg2+-ion binding sites within SLVI. Four Mg2+ ions in SLVI are associated with known RNA structural motifs, including the G−U wobble pair and the GNRA tetraloop, and our studies reveal novel insights about Mg2+ ion binding to these RNA motifs. Interestingly, one Mg2+ ion is specifically associated with the S-turn motif, confirming its structural role in the folding of the A730 loop. This Mg2+ ion is likely important for formation of the active site and may play an indirec
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Rational engineering of the Neurospora VS Ribozyme to allow substrate recognition via different kissing-loop interactions
Nucleic acids research, 2016Co-Authors: Julie Lacroix-labonté, Nicolas Girard, Pierre Dagenais, Pascale LegaultAbstract:The Neurospora VS Ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS Ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS Ribozyme. Next, substrate/Ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS Ribozyme can be adapted to cleave other folded RNA substrates.
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The NMR structure of the II-III-VI three-way junction from the Neurospora VS Ribozyme reveals a critical tertiary interaction and provides new insights into the global Ribozyme structure.
RNA (New York N.Y.), 2015Co-Authors: Eric Bonneau, Nicolas Girard, Sébastien Lemieux, Pascale LegaultAbstract:As part of an effort to structurally characterize the complete Neurospora VS Ribozyme, NMR solution structures of several subdomains have been previously determined, including the internal loops of domains I and VI, the I/V kissing-loop interaction and the III-IV-V junction. Here, we expand this work by determining the NMR structure of a 62-nucleotide RNA (J236) that encompasses the VS Ribozyme II-III-VI three-way junction and its adjoining stems. In addition, we localize Mg(2+)-binding sites within this structure using Mn(2+)-induced paramagnetic relaxation enhancement. The NMR structure of the J236 RNA displays a family C topology with a compact core stabilized by continuous stacking of stems II and III, a cis WC/WC G•A base pair, two base triples and two Mg(2+) ions. Moreover, it reveals a remote tertiary interaction between the adenine bulges of stems II and VI. Additional NMR studies demonstrate that both this bulge-bulge interaction and Mg(2+) ions are critical for the stable folding of the II-III-VI junction. The NMR structure of the J236 RNA is consistent with biochemical studies on the complete VS Ribozyme, but not with biophysical studies performed with a minimal II-III-VI junction that does not contain the II-VI bulge-bulge interaction. Together with previous NMR studies, our findings provide important new insights into the three-dimensional architecture of this unique Ribozyme.
David M J Lilley - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of the Varkud satellite Ribozyme
Nature Chemical Biology, 2015Co-Authors: Nikolai B Suslov, David M J Lilley, Saurja Dasgupta, Hao Huang, James R Fuller, Phoebe A Rice, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this Ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans . This mode of RNA−RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead Ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution. Crystal structures of the full-length VS Ribozyme show a domain-swapped dimer that reveals potential mechanisms for cis and trans processing, and suggest convergent evolution in the active site motifs across multiple Ribozymes.
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Crystal structure of the Varkud satellite Ribozyme
Nature chemical biology, 2015Co-Authors: Nikolai B Suslov, David M J Lilley, Saurja Dasgupta, Hao Huang, James R Fuller, Phoebe A Rice, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this Ribozyme from Neurospora intermedia at 3.1 A resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead Ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.
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synthesis of novel tetrazole c5 linked c0 and c2 ribonucleoside phosphoramidites using mepom and pom groups for probing rna catalysis
Tetrahedron Letters, 2012Co-Authors: Shinya Harusawa, Zheng-yun Zhao, Timothy J. Wilson, Hiroki Yoneyama, Daiki Fujisue, Masayoshi Nishiura, Mihoyo Fujitake, Yoshihide Usami, Scott A Mcphee, David M J LilleyAbstract:Novel C5-linked C0- and C2-tetrazole ribonucleoside phosphoramidites were designed and synthesized via tetrazole C-nucleosides. Pivaloyloxymethyl (POM) and methyl-substituted POM (MePOM) groups were introduced as N-protecting groups in the tetrazole ring that can be readily removed under mild basic conditions. The phosphoramidites were successfully incorporated into the VS Ribozyme substrate and hence providing a chemogenetic approach to determine which nucleobases of Ribozymes function as the acid or base, in the studies of Ribozyme general acid and base catalysis.
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Do the hairpin and VS Ribozymes share a common catalytic mechanism based on general acid-base catalysis? A critical assessment of available experimental data.
RNA (New York N.Y.), 2010Co-Authors: Timothy J. Wilson, David M J LilleyAbstract:The active centers of the hairpin and VS Ribozymes are both generated by the interaction of two internal loops, and both Ribozymes use guanine and adenine nucleobases to accelerate cleavage and ligation reactions. The centers are topologically equivalent and the relative positioning of key elements the same. There is good evidence that the cleavage reaction of the VS Ribozyme is catalyzed by the guanine (G638) acting as general base and the adenine (A756) as general acid. We now critically evaluate the experimental mechanistic evidence for the hairpin Ribozyme. We conclude that all the available data are fully consistent with a major contribution to catalysis by general acid–base catalysis involving the adenine (A38) and guanine (G8). It appears that the two Ribozymes are mechanistically equivalent.
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Nucleobase-mediated general acid-base catalysis in the Varkud satellite Ribozyme.
Proceedings of the National Academy of Sciences of the United States of America, 2010Co-Authors: Timothy J. Wilson, John K. Frederiksen, Nan-sheng Li, Joseph A. Piccirilli, Jun Lu, David M J LilleyAbstract:Existing evidence suggests that the Varkud satellite (VS) Ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the Ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pKa = 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G Ribozyme, consistent with general acid catalysis by A756 in the unmodified Ribozyme. The pH dependence of the cleavage of the phosphorothiolate-modified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS Ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756.
Joseph A. Piccirilli - One of the best experts on this subject based on the ideXlab platform.
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The L-platform/L-scaffold framework: a blueprint for RNA-cleaving nucleic acid enzyme design
RNA (New York N.Y.), 2019Co-Authors: Colin S. Gaines, Joseph A. Piccirilli, Darrin M. YorkAbstract:We develop an L-platform/L-scaffold framework we hypothesize may serve as a blueprint to facilitate site-specific RNA-cleaving nucleic acid enzyme design. Building on the L-platform motif originally described by Suslov and coworkers, we identify new critical scaffolding elements required to anchor a conserved general base guanine ("L-anchor") and bind functionally important metal ions at the active site ("L-pocket"). Molecular simulations, together with a broad range of experimental structural and functional data, connect the L-platform/L-scaffold elements to necessary and sufficient conditions for catalytic activity. We demonstrate that the L-platform/L-scaffold framework is common to five of the nine currently known naturally occurring Ribozyme classes (Twr, HPr, VSr, HHr, Psr), and intriguingly from a design perspective, the framework also appears in an artificially engineered DNAzyme (8-17dz). The flexibility of the L-platform/L-scaffold framework is illustrated on these systems, highlighting modularity and trends in the variety of known general acid moieties that are supported. These trends give rise to two distinct catalytic paradigms, building on the classifications proposed by Wilson and coworkers and named for the implicated general base and acid. The "G + A" paradigm (Twr, HPr, VSr) exclusively utilizes nucleobase residues for chemistry, and the "G + M + " paradigm (HHr, 8-17dz, Psr) involves structuring of the "L-pocket" metal ion binding site for recruitment of a divalent metal ion that plays an active role in the chemical steps of the reaction. Finally, the modularity of the L-platform/L-scaffold framework is illustrated in the VS Ribozyme where the "L-pocket" assumes the functional role of the "L-anchor" element, highlighting a distinct mechanism, but one that is functionally linked with the hammerhead Ribozyme.
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Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme.
Journal of the American Chemical Society, 2017Co-Authors: Saurja Dasgupta, Nikolai B Suslov, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the Ribozyme, resulting in a rearrangement of the substrate helix register into a so-called “shifted” conformation that is critical for substrate binding and activation. We report a 3.3 A crystal structure of the complete Ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the Ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin Ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and Ribozyme cleavage.
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Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme
2017Co-Authors: Saurja Dasgupta, Nikolai B Suslov, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the Ribozyme, resulting in a rearrangement of the substrate helix register into a so-called “shifted” conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete Ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the Ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin Ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and Ribozyme cleavage
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Crystal structure of the Varkud satellite Ribozyme
Nature Chemical Biology, 2015Co-Authors: Nikolai B Suslov, David M J Lilley, Saurja Dasgupta, Hao Huang, James R Fuller, Phoebe A Rice, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this Ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans . This mode of RNA−RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead Ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution. Crystal structures of the full-length VS Ribozyme show a domain-swapped dimer that reveals potential mechanisms for cis and trans processing, and suggest convergent evolution in the active site motifs across multiple Ribozymes.
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Crystal structure of the Varkud satellite Ribozyme
Nature chemical biology, 2015Co-Authors: Nikolai B Suslov, David M J Lilley, Saurja Dasgupta, Hao Huang, James R Fuller, Phoebe A Rice, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this Ribozyme from Neurospora intermedia at 3.1 A resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead Ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.
Saurja Dasgupta - One of the best experts on this subject based on the ideXlab platform.
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Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme.
Journal of the American Chemical Society, 2017Co-Authors: Saurja Dasgupta, Nikolai B Suslov, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the Ribozyme, resulting in a rearrangement of the substrate helix register into a so-called “shifted” conformation that is critical for substrate binding and activation. We report a 3.3 A crystal structure of the complete Ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the Ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin Ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and Ribozyme cleavage.
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Structural Basis for Substrate Helix Remodeling and Cleavage Loop Activation in the Varkud Satellite Ribozyme
2017Co-Authors: Saurja Dasgupta, Nikolai B Suslov, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme catalyzes site-specific RNA cleavage and ligation reactions. Recognition of the substrate involves a kissing loop interaction between the substrate and the catalytic domain of the Ribozyme, resulting in a rearrangement of the substrate helix register into a so-called “shifted” conformation that is critical for substrate binding and activation. We report a 3.3 Å crystal structure of the complete Ribozyme that reveals the active, shifted conformation of the substrate, docked into the catalytic domain of the Ribozyme. Comparison to previous NMR structures of isolated, inactive substrates provides a physical description of substrate remodeling, and implicates roles for tertiary interactions in catalytic activation of the cleavage loop. Similarities to the hairpin Ribozyme cleavage loop activation suggest general strategies to enhance fidelity in RNA folding and Ribozyme cleavage
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Crystal structure of the Varkud satellite Ribozyme
Nature Chemical Biology, 2015Co-Authors: Nikolai B Suslov, David M J Lilley, Saurja Dasgupta, Hao Huang, James R Fuller, Phoebe A Rice, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this Ribozyme from Neurospora intermedia at 3.1 Å resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans . This mode of RNA−RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead Ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution. Crystal structures of the full-length VS Ribozyme show a domain-swapped dimer that reveals potential mechanisms for cis and trans processing, and suggest convergent evolution in the active site motifs across multiple Ribozymes.
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Crystal structure of the Varkud satellite Ribozyme
Nature chemical biology, 2015Co-Authors: Nikolai B Suslov, David M J Lilley, Saurja Dasgupta, Hao Huang, James R Fuller, Phoebe A Rice, Joseph A. PiccirilliAbstract:The Varkud satellite (VS) Ribozyme mediates rolling-circle replication of a plasmid found in the Neurospora mitochondrion. We report crystal structures of this Ribozyme from Neurospora intermedia at 3.1 A resolution, which revealed an intertwined dimer formed by an exchange of substrate helices. In each protomer, an arrangement of three-way helical junctions organizes seven helices into a global fold that creates a docking site for the substrate helix of the other protomer, resulting in the formation of two active sites in trans. This mode of RNA-RNA association resembles the process of domain swapping in proteins and has implications for RNA regulation and evolution. Within each active site, adenine and guanine nucleobases abut the scissile phosphate, poised to serve direct roles in catalysis. Similarities to the active sites of the hairpin and hammerhead Ribozymes highlight the functional importance of active-site features, underscore the ability of RNA to access functional architectures from distant regions of sequence space, and suggest convergent evolution.
