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The global structure of the VS ribozyme.

ABSTRACT: The VS ribozyme comprises five helical segments (II-VI) in a formal H shape, organized by two three-way junctions. It interacts with its stem-loop substrate (I) by tertiary interactions. We have determined the global shape of the 3-4-5 junction (relating helices III-V) by electrophoresis and FRET. Estimation of the dihedral angle between helices II and V electrophoretically has allowed us to build a model for the global structure of the complete ribozyme. We propose that the substrate is docked into a cleft between helices II and VI, with its loop making a tertiary interaction with that of helix V. This is consistent with the dependence of activity on the length of helix III. The scissile phosphate is well placed to interact with the probable active site of the ribozyme, the loop containing A730.

SUBMITTER: Lafontaine DA

PROVIDER: S-EPMC126006 | biostudies-literature | 2002 May

REPOSITORIES: biostudies-literature

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The global structure of the VS ribozyme.

Lafontaine Daniel A DA Norman David G DG Lilley David M J DM

The EMBO journal 20020501 10

The VS ribozyme comprises five helical segments (II-VI) in a formal H shape, organized by two three-way junctions. It interacts with its stem-loop substrate (I) by tertiary interactions. We have determined the global shape of the 3-4-5 junction (relating helices III-V) by electrophoresis and FRET. Estimation of the dihedral angle between helices II and V electrophoretically has allowed us to build a model for the global structure of the complete ribozyme. We propose that the substrate is docked ...[more]

PMID: 12006498

Similar Datasets

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.

Project description: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.

| S-EPMC4536322 | biostudies-literature

Structure of the ribozyme substrate hairpin of Neurospora VS RNA: a close look at the cleavage site.

Project description:The cleavage site of the Neurospora VS RNA ribozyme is located in a separate hairpin domain containing a hexanucleotide internal loop with an A-C mismatch and two adjacent G-A mismatches. The solution structure of the internal loop and helix la of the ribozyme substrate hairpin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The 2 nt in the internal loop, flanking the cleavage site, a guanine and adenine, are involved in two sheared G.A base pairs similar to the magnesium ion-binding site of the hammerhead ribozyme. Adjacent to the tandem G.A base pairs, the adenine and cytidine, which are important for cleavage, form a noncanonical wobble A+-C base pair. The dynamic properties of the internal loop and details of the high-resolution structure support the view that the hairpin structure represents a ground state, which has to undergo a conformational change prior to cleavage. Results of chemical modification and mutagenesis data of the Neurospora VS RNA ribozyme can be explained in context with the present three-dimensional structure.

| S-EPMC1370051 | biostudies-other

NMR structure of the A730 loop of the Neurospora VS ribozyme: insights into the formation of the active site.

Project description:The Neurospora VS ribozyme is a small nucleolytic ribozyme with unique primary, secondary and global tertiary structures, which displays mechanistic similarities to the hairpin ribozyme. Here, we determined the high-resolution NMR structure of a stem-loop VI fragment containing the A730 internal loop, which forms part of the active site. In the presence of magnesium ions, the A730 loop adopts a structure that is consistent with existing biochemical data and most likely reflects its conformation in the VS ribozyme prior to docking with the cleavage site internal loop. Interestingly, the A730 loop adopts an S-turn motif that is also present in loop B within the hairpin ribozyme active site. The S-turn appears necessary to expose the Watson-Crick edge of a catalytically important residue (A756) so that it can fulfill its role in catalysis. The A730 loop and the cleavage site loop of the VS ribozyme display structural similarities to internal loops found in the active site of the hairpin ribozyme. These similarities provided a rationale to build a model of the VS ribozyme active site based on the crystal structure of the hairpin ribozyme.

| S-EPMC3105416 | biostudies-literature

Solvent structure and hammerhead ribozyme catalysis.

Project description:Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 A structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn(2+) is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2'-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.

| S-EPMC2610685 | biostudies-literature

Single VS ribozyme molecules reveal dynamic and hierarchical folding toward catalysis.

Project description: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.

| S-EPMC2575853 | biostudies-literature

Structure and mechanism of a methyltransferase ribozyme.

Project description:Known ribozymes in contemporary biology perform a limited range of chemical catalysis, but in vitro selection has generated species that catalyze a broader range of chemistry; yet, there have been few structural and mechanistic studies of selected ribozymes. A ribozyme has recently been selected that can catalyze a site-specific methyl transfer reaction. We have solved the crystal structure of this ribozyme at a resolution of 2.3 Å, showing how the RNA folds to generate a very specific binding site for the methyl donor substrate. The structure immediately suggests a catalytic mechanism involving a combination of proximity and orientation and nucleobase-mediated general acid catalysis. The mechanism is supported by the pH dependence of the rate of catalysis. A selected methyltransferase ribozyme can thus use a relatively sophisticated catalytic mechanism, broadening the range of known RNA-catalyzed chemistry.

| S-EPMC9050513 | biostudies-literature

Crystal structure of the Varkud satellite ribozyme.

Project description: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.

| S-EPMC4618023 | biostudies-literature

Role of SLV in SLI substrate recognition by the Neurospora VS ribozyme.

Project description:Substrate recognition by the VS ribozyme involves a magnesium-dependent loop/loop interaction between the SLI substrate and the SLV hairpin from the catalytic domain. Recent NMR studies of SLV demonstrated that magnesium ions stabilize a U-turn loop structure and trigger a conformational change for the extruded loop residue U700, suggesting a role for U700 in SLI recognition. Here, we kinetically characterized VS ribozyme mutants to evaluate the contribution of U700 and other SLV loop residues to SLI recognition. To help interpret the kinetic data, we structurally characterized the SLV mutants by NMR spectroscopy and generated a three-dimensional model of the SLI/SLV complex by homology modeling with MC-Sym. We demonstrated that the mutation of U700 by A, C, or G does not significantly affect ribozyme activity, whereas deletion of U700 dramatically impairs this activity. The U700 backbone is likely important for SLI recognition, but does not appear to be required for either the structural integrity of the SLV loop or for direct interactions with SLI. Thus, deletion of U700 may affect other aspects of SLI recognition, such as magnesium ion binding and SLV loop dynamics. As part of our NMR studies, we developed a convenient assay based on detection of unusual (31)P and (15)N N7 chemical shifts to probe the formation of U-turn structures in RNAs. Our model of the SLI/SLV complex, which is compatible with biochemical data, leads us to propose novel interactions at the loop I/loop V interface.

| S-EPMC2271362 | biostudies-literature

Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture.

Project description:Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure-function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing-loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (k(cat)/K(M)) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

| S-EPMC3299992 | biostudies-literature

The Structure of Adamantane Clusters: Atomistic vs. Coarse-Grained Predictions From Global Optimization.

Project description:Candidate structures for the global minima of adamantane clusters, (C10H16) N , are presented. Based on a rigid model for individual molecules with atom-atom pairwise interactions that include Lennard-Jones and Coulomb contributions, low-energy structures were obtained up to N = 42 using the basin-hopping method. The results indicate that adamantane clusters initially grow accordingly with an icosahedral packing scheme, followed above N = 14 by a structural transition toward face-centered cubic structures. The special stabilities obtained at N = 13, 19, and 38 are consistent with these two structural families, and agree with recent mass spectrometry measurements on cationic adamantane clusters. Coarse-graining the intermolecular potential by averaging over all possible orientations only partially confirm the all-atom results, the magic numbers at 13 and 38 being preserved. However, the details near the structural transition are not captured well, because despite their high symmetry the adamantane molecules are still rather anisotropic.

| S-EPMC6707085 | biostudies-literature

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