RESUMEN
RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.
Asunto(s)
Aptámeros de Nucleótidos/química , ARN Catalítico/química , ARN/química , Secuencia de Bases , Ligandos , Conformación de Ácido Nucleico , Riboswitch/genéticaRESUMEN
The hammerhead ribozyme is a well-studied nucleolytic ribozyme that catalyzes the self-cleavage of the RNA phosphodiester backbone. Despite experimental and theoretical efforts, key questions remain about details of the mechanism with regard to the activation of the nucleophile by the putative general base guanine (G12). Straightforward interpretation of the measured activity-pH data implies the pKa value of the N1 position in the G12 nucleobase is significantly shifted by the ribozyme environment. Recent crystallographic and biochemical work has identified pH-dependent divalent metal ion binding at the N7/O6 position of G12, leading to the hypothesis that this binding mode could induce a pKa shift of G12 toward neutrality. We present computational results that support this hypothesis and provide a model that unifies the interpretation of available structural and biochemical data and paints a detailed mechanistic picture of the general base step of the reaction. Experimentally testable predictions are made for mutational and rescue effects on G12, which will give further insights into the catalytic mechanism. These results contribute to our growing knowledge of the potential roles of divalent metal ions in RNA catalysis.
Asunto(s)
Biocatálisis , Cationes Bivalentes/metabolismo , Guanina/metabolismo , Metales/metabolismo , ARN Catalítico/química , ARN Catalítico/metabolismo , Biocatálisis/efectos de los fármacos , Cationes Bivalentes/química , Cationes Bivalentes/farmacología , Guanina/química , Metales/química , Metales/farmacología , Simulación de Dinámica Molecular , Teoría Cuántica , TermodinámicaRESUMEN
The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the pKA of G12. On the basis of this crystal structure as well as a wealth of biochemical studies, we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.
Asunto(s)
Magnesio/química , Conformación de Ácido Nucleico , ARN Catalítico/química , Cristalografía por Rayos XRESUMEN
The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.
Asunto(s)
Magnesio/metabolismo , Manganeso/metabolismo , ARN Catalítico/metabolismo , ARN de Helminto/metabolismo , Schistosoma mansoni/enzimología , Zinc/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Cationes Bivalentes/metabolismo , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Mutación Puntual , Protones , ARN Catalítico/química , ARN Catalítico/genética , ARN de Helminto/química , ARN de Helminto/genética , Schistosoma mansoni/química , Schistosoma mansoni/metabolismo , Esquistosomiasis mansoni/parasitología , Especificidad por SustratoRESUMEN
PICKLE plays a critical role in repression of genes that regulate development identity in Arabidopsis thaliana. PICKLE codes for a putative ATP-dependent chromatin remodeler that exhibits sequence similarity to members of subfamily II of animal CHD remodelers, which includes remodelers such as CHD3/Mi-2 that also restrict expression of developmental regulators. Whereas animal CHD3 remodelers are a component of the Mi-2/NuRD complex that promotes histone deacetylation, PICKLE promotes trimethylation of histone H3 lysine 27 suggesting that it acts via a distinct epigenetic pathway. Here, we examine whether PICKLE is also a member of a multisubunit complex and characterize the biochemical properties of recombinant PICKLE protein. Phylogenetic analysis indicates that PICKLE-related proteins in plants share a common ancestor with members of subfamily II of animal CHD remodelers. Biochemical characterization of PICKLE in planta, however, reveals that PICKLE primarily exists as a monomer. Recombinant PICKLE protein is an ATPase that is stimulated by ssDNA and mononucleosomes and binds to both naked DNA and mononucleosomes. Furthermore, recombinant PICKLE exhibits ATP-dependent chromatin remodeling activity. These studies demonstrate that subfamily II CHD proteins in plants, such as PICKLE, retain ATP-dependent chromatin remodeling activity but act through a mechanism that does not involve the ubiquitous Mi-2/NuRD complex.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , ADN Helicasas , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/genética , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Histonas/genética , Lisina/genética , Lisina/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Datos de Secuencia Molecular , FilogeniaRESUMEN
The 'RNA world' hypothesis holds that during evolution the structural and enzymatic functions initially served by RNA were assumed by proteins, leading to the latter's domination of biological catalysis. This progression can still be seen in modern biology, where ribozymes, such as the ribosome and RNase P, have evolved into protein-dependent RNA catalysts ('RNPzymes'). Similarly, group I introns use RNA-catalysed splicing reactions, but many function as RNPzymes bound to proteins that stabilize their catalytically active RNA structure. One such protein, the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (TyrRS; CYT-18), is bifunctional and both aminoacylates mitochondrial tRNA(Tyr) and promotes the splicing of mitochondrial group I introns. Here we determine a 4.5-A co-crystal structure of the Twort orf142-I2 group I intron ribozyme bound to splicing-active, carboxy-terminally truncated CYT-18. The structure shows that the group I intron binds across the two subunits of the homodimeric protein with a newly evolved RNA-binding surface distinct from that which binds tRNA(Tyr). This RNA binding surface provides an extended scaffold for the phosphodiester backbone of the conserved catalytic core of the intron RNA, allowing the protein to promote the splicing of a wide variety of group I introns. The group I intron-binding surface includes three small insertions and additional structural adaptations relative to non-splicing bacterial TyrRSs, indicating a multistep adaptation for splicing function. The co-crystal structure provides insight into how CYT-18 promotes group I intron splicing, how it evolved to have this function, and how proteins could have incrementally replaced RNA structures during the transition from an RNA world to an RNP world.
Asunto(s)
Intrones/genética , Neurospora crassa/enzimología , Empalme del ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Tirosina-ARNt Ligasa/química , Tirosina-ARNt Ligasa/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Conformación Molecular , Unión Proteica , ARN/genética , ARN/metabolismo , ARN Catalítico/química , ARN Catalítico/genética , ARN Catalítico/metabolismo , Fagos de Staphylococcus/enzimología , Fagos de Staphylococcus/genéticaRESUMEN
The hepatitis delta virus ribozyme catalyzes an RNA cleavage reaction using a catalytic nucleobase and a divalent metal ion. The catalytic base, C75, serves as a general acid and has a pK(a) shifted toward neutrality. Less is known about the role of metal ions in the mechanism. A recent crystal structure of the precleavage ribozyme identified a Mg²âº ion that interacts through its partial hydration sphere with the G25·U20 reverse wobble. In addition, this Mg²âº ion is in position to directly coordinate the nucleophile, the 2'-hydroxyl of U(-1), suggesting it can serve as a Lewis acid to facilitate deprotonation of the 2'-hydroxyl. To test the role of the active site Mg²âº ion, we replaced the G25·U20 reverse wobble with an isosteric A25·C20 reverse wobble. This change was found to significantly reduce the negative potential at the active site, as supported by electrostatics calculations, suggesting that active site Mg²âº binding could be adversely affected by the mutation. The kinetic analysis and molecular dynamics of the A25·C20 double mutant suggest that this variant stably folds into an active structure. However, pH-rate profiles of the double mutant in the presence of Mg²âº are inverted relative to the profiles for the wild-type ribozyme, suggesting that the A25·C20 double mutant has lost the active site metal ion. Overall, these studies support a model in which the partially hydrated Mg²âº positioned at the G25·U20 reverse wobble is catalytic and could serve as a Lewis acid, a Brønsted base, or both to facilitate deprotonation of the nucleophile.
Asunto(s)
Virus de la Hepatitis Delta/metabolismo , Magnesio/química , ARN Catalítico/metabolismo , ARN Viral/metabolismo , Biocatálisis , Dominio Catalítico , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Magnesio/metabolismo , Simulación de Dinámica Molecular , Mutación , Pliegue del ARN , Estabilidad del ARN , ARN Catalítico/química , ARN Viral/química , Electricidad Estática , Propiedades de Superficie , Agua/análisisRESUMEN
Metal ion and nucleobase catalysis are important for ribozyme mechanism, but the extent to which they cooperate is unclear. A crystal structure of the hepatitis delta virus (HDV) ribozyme suggested that the pro-RP oxygen at the scissile phosphate directly coordinates a catalytic Mg(2+) ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies of the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn(2+). Here, we combine experiment and theory to explore phosphorothioate substitutions at the scissile phosphate. We report significant thio effects at the scissile phosphate and metal ion rescue with Cd(2+). Reaction profiles with an SP-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg(2+) or Cd(2+), supporting the idea that the pro-SP oxygen does not coordinate metal ions. The RP-phosphorothioate substitution, however, exhibits biphasic kinetics, with the fast-reacting phase displaying a thio effect of up to 5-fold and the slow-reacting phase displaying a thio effect of ~1000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd(2+) of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd(2+) inhibiting the oxo substrate but not the RP substrate. This metal ion rescue suggests a direct interaction of the catalytic metal ion with the pro-RP oxygen, in line with experiments with the antigenomic HDV ribozyme. Experiments without divalent ions, with a double mutant that interferes with Mg(2+) binding, or with C75 deleted suggest that the pro-RP oxygen plays at most a redundant role in positioning C75. Quantum mechanical/molecular mechanical (QM/MM) studies indicate that the metal ion contributes to catalysis by interacting with both the pro-RP oxygen and the nucleophilic 2'-hydroxyl, supporting the experimental findings.
Asunto(s)
Cadmio/química , Virus de la Hepatitis Delta/genética , Magnesio/química , Organotiofosfatos/química , Oxígeno/química , ARN Catalítico/genética , Azufre/química , Catálisis , Dominio Catalítico , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Teoría Cuántica , ARN Catalítico/química , ARN Catalítico/metabolismoRESUMEN
Functional and kinetic constraints must be efficiently balanced during the folding process of all biopolymers. To understand how homologous RNA molecules with different global architectures fold into a common core structure we determined, under identical conditions, the folding mechanisms of three phylogenetically divergent group I intron ribozymes. These ribozymes share a conserved functional core defined by topologically equivalent tertiary motifs but differ in their primary sequence, size, and structural complexity. Time-resolved hydroxyl radical probing of the backbone solvent accessible surface and catalytic activity measurements integrated with structural-kinetic modeling reveal that each ribozyme adopts a unique strategy to attain the conserved functional fold. The folding rates are not dictated by the size or the overall structural complexity, but rather by the strength of the constituent tertiary motifs which, in turn, govern the structure, stability, and lifetime of the folding intermediates. A fundamental general principle of RNA folding emerges from this study: The dominant folding flux always proceeds through an optimally structured kinetic intermediate that has sufficient stability to act as a nucleating scaffold while retaining enough conformational freedom to avoid kinetic trapping. Our results also suggest a potential role of naturally selected peripheral A-minor interactions in balancing RNA structural stability with folding efficiency.
Asunto(s)
Bacteriófagos/enzimología , Dominio Catalítico , Pliegue de Proteína , ARN Catalítico/química , Rhodocyclaceae/enzimología , Tetrahymena/enzimología , Secuencia de Bases , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Catalítico/metabolismo , Homología Estructural de ProteínaRESUMEN
Although RNA molecules are highly negatively charged, anions have been observed bound to RNA in crystal structures. It has been proposed that anion binding sites found within isolated RNAs represent regions of the molecule that could be involved in intermolecular interactions, indicating potential contact points for negatively charged amino acids from proteins or phosphate groups from an RNA. Several types of anion binding sites have been cataloged based on available structures. However, currently there is no method for unambiguously assigning anions to crystallographic electron density, and this has precluded more detailed analysis of RNA-anion interaction motifs and their significance. We therefore soaked selenate into two different types of RNA crystals and used the anomalous signal from these anions to identify binding sites in these RNA molecules unambiguously. Examination of these sites and comparison with other suspected anion binding sites reveals features of anion binding motifs, and shows that selenate may be a useful tool for studying RNA-anion interactions.
Asunto(s)
Aniones/metabolismo , ARN/química , ARN/metabolismo , Aminoácidos/metabolismo , Aniones/química , Sitios de Unión , Cationes/química , Cationes/metabolismo , Cristalografía , Modelos Moleculares , Conformación de Ácido Nucleico , Proteínas/química , Proteínas/metabolismo , Ácido Selénico , Compuestos de Selenio/química , Compuestos de Selenio/metabolismo , Sulfatos/química , Sulfatos/metabolismo , Difracción de Rayos XRESUMEN
The hepatitis delta virus (HDV) ribozyme and related RNAs are widely dispersed in nature. This RNA is a small nucleolytic ribozyme that self-cleaves to generate products with a 2',3'-cyclic phosphate and a free 5'-hydroxyl. Although small ribozymes are dependent on divalent metal ions under biologically relevant buffer conditions, they function in the absence of divalent metal ions at high ionic strengths. This characteristic suggests that a functional group within the covalent structure of small ribozymes is facilitating catalysis. Structural and mechanistic analyses have demonstrated that the HDV ribozyme active site contains a cytosine with a perturbed pK(a) that serves as a general acid to protonate the leaving group. The reaction of the HDV ribozyme in monovalent cations alone never approaches the velocity of the Mg(2+)-dependent reaction, and there is significant biochemical evidence that a Mg(2+) ion participates directly in catalysis. A recent crystal structure of the HDV ribozyme revealed that there is a metal binding pocket in the HDV ribozyme active site. Modeling of the cleavage site into the structure suggested that this metal ion can interact directly with the scissile phosphate and the nucleophile. In this manner, the Mg(2+) ion can serve as a Lewis acid, facilitating deprotonation of the nucleophile and stabilizing the conformation of the cleavage site for in-line attack of the nucleophile at the scissile phosphate. This catalytic strategy had previously been observed only in much larger ribozymes. Thus, in contrast to most large and small ribozymes, the HDV ribozyme uses two distinct catalytic strategies in its cleavage reaction.
Asunto(s)
Virus de la Hepatitis Delta/enzimología , Modelos Moleculares , ARN Catalítico/química , ARN Viral/química , Ribonucleoproteína Nuclear Pequeña U1/química , Animales , Dominio Catalítico , Cristalografía por Rayos X , Nucleótidos de Citosina/química , Estabilidad de Enzimas , Humanos , Magnesio/química , ARN Catalítico/metabolismo , ARN Viral/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Especificidad por SustratoRESUMEN
The hepatitis delta virus (HDV) ribozyme uses both metal ion and nucleobase catalysis in its cleavage mechanism. A reverse G·U wobble was observed in a recent crystal structure of the precleaved state. This unusual base pair positions a Mg(2+) ion to participate in catalysis. Herein, we used molecular dynamics (MD) and X-ray crystallography to characterize the conformation and metal binding characteristics of this base pair in product and precleaved forms. Beginning with a crystal structure of the product form, we observed formation of the reverse G·U wobble during MD trajectories. We also demonstrated that this base pair is compatible with the diffraction data for the product-bound state. During MD trajectories of the product form, Na(+) ions interacted with the reverse G·U wobble in the RNA active site, and a Mg(2+) ion, introduced in certain trajectories, remained bound at this site. Beginning with a crystal structure of the precleaved form, the reverse G·U wobble with bound Mg(2+) remained intact during MD simulations. When we removed Mg(2+) from the starting precleaved structure, Na(+) ions interacted with the reverse G·U wobble. In support of the computational results, we observed competition between Na(+) and Mg(2+) in the precleaved ribozyme crystallographically. Nonlinear Poisson-Boltzmann calculations revealed a negatively charged patch near the reverse G·U wobble. This anionic pocket likely serves to bind metal ions and to help shift the pK(a) of the catalytic nucleobase, C75. Thus, the reverse G·U wobble motif serves to organize two catalytic elements, a metal ion and catalytic nucleobase, within the active site of the HDV ribozyme.
Asunto(s)
Dominio Catalítico , Virus de la Hepatitis Delta/metabolismo , Magnesio/metabolismo , Dominios y Motivos de Interacción de Proteínas , ARN Catalítico/química , ARN Catalítico/metabolismo , Sodio/metabolismo , Unión Competitiva , Biocatálisis , Bases de Datos de Ácidos Nucleicos , Cinética , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Distribución de Poisson , Propiedades de SuperficieRESUMEN
The hepatitis delta virus (HDV) ribozyme and HDV-like ribozymes are self-cleaving RNAs found throughout all kingdoms of life. These RNAs fold into a double-nested pseudoknot structure and cleave RNA, yielding 2',3'-cyclic phosphate and 5'-hydroxyl termini. The active site nucleotide C75 has a pK(a) shifted >2 pH units toward neutrality and has been implicated as a general acid/base in the cleavage reaction. An active site Mg(2+) ion that helps activate the 2'-hydroxyl for nucleophilic attack has been characterized biochemically; however, this ion has not been visualized in any previous structures. To create a snapshot of the ribozyme in a state poised for catalysis, we have crystallized and determined the structure of the HDV ribozyme bound to an inhibitor RNA containing a deoxynucleotide at the cleavage site. This structure includes the wild-type C75 nucleotide and Mg(2+) ions, both of which are required for maximal ribozyme activity. This structure suggests that the position of C75 does not change during the cleavage reaction. A partially hydrated Mg(2+) ion is also found within the active site where it interacts with a newly resolved G.U reverse wobble. Although the inhibitor exhibits crystallographic disorder, we modeled the ribozyme-substrate complex using the conformation of the inhibitor strand observed in the hammerhead ribozyme. This model suggests that the pro-R(P) oxygen of the scissile phosphate and the 2'-hydroxyl nucleophile are inner-sphere ligands to the active site Mg(2+) ion. Thus, the HDV ribozyme may use a combination of metal ion Lewis acid and nucleobase general acid strategies to effect RNA cleavage.
Asunto(s)
Virus de la Hepatitis Delta/enzimología , ARN Catalítico/química , Dominio Catalítico , Cristalografía por Rayos X , Hidrólisis , Magnesio , Fenómenos Químicos Orgánicos , Organofosfatos/metabolismo , ARN Catalítico/metabolismoRESUMEN
The hepatitis delta virus (HDV) ribozyme occurs in the genomic and antigenomic strands of the HDV RNA and within mammalian transcriptomes. Previous kinetic studies suggested that a wobble pair (G*U or A(+)*C) is preferred at the cleavage site; however, the reasons for this are unclear. We conducted sequence comparisons, which indicated that while G*U is the most prevalent combination at the cleavage site, G-C occurs to a significant extent in genomic HDV isolates, and G*U, G-C, and A-U pairs are present in mammalian ribozymes. We analyzed the folding of genomic HDV ribozymes by free energy minimization and found that variants with purine-pyrimidine combinations at the cleavage site are predicted to form native structures while pyrimidine-purine combinations misfold, consistent with earlier kinetic data and sequence comparisons. To test whether the cleavage site base pair contributes to catalysis, we characterized the pH and Mg(2+)-dependence of reaction kinetics of fast-folding genomic HDV ribozymes with cleavage site base pair purine-pyrimidine combinations: G*U, A-U, G-C, and A(+)*C. Rates for these native-folding ribozymes displayed highly similar pH and Mg(2+) concentration dependencies, with the exception of the A(+)*C ribozyme, which deviated at high pH. None of the four ribozymes underwent miscleavage. These observations support the A(+)*C ribozyme as being more active with a wobble pair at the cleavage site than with no base pair at all. Overall, the data support a model in which the cleavage site base pair provides a structural role in catalysis and does not need to be a wobble pair.
Asunto(s)
Emparejamiento Base , Virus de la Hepatitis Delta/enzimología , Virus de la Hepatitis Delta/genética , ARN Catalítico/metabolismo , ARN Viral/metabolismo , Secuencia de Bases , Catálisis , Genoma Viral , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/genética , ARN Viral/química , ARN Viral/genéticaRESUMEN
Group I introns are catalytic RNAs capable of orchestrating two sequential phosphotransesterification reactions that result in self-splicing. To understand how the group I intron active site facilitates catalysis, we have solved the structure of an active ribozyme derived from the orf142-I2 intron from phage Twort bound to a four-nucleotide product RNA at a resolution of 3.6 A. In addition to the three conserved domains characteristic of all group I introns, the Twort ribozyme has peripheral insertions characteristic of phage introns. These elements form a ring that completely envelops the active site, where a snug pocket for guanosine is formed by a series of stacked base triples. The structure of the active site reveals three potential binding sites for catalytic metals, and invokes a role for the 2' hydroxyl of the guanosine substrate in organization of the active site for catalysis.
Asunto(s)
Bacteriófagos/enzimología , Bacteriófagos/genética , Intrones/genética , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/metabolismo , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Guanosina/química , Guanosina/metabolismo , Metales/química , Metales/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatos/metabolismo , ARN Catalítico/genéticaRESUMEN
Raman crystallography is the application of Raman spectroscopy to single crystals. This technique has been applied to a variety of protein molecules where it has provided unique information about biopolymer folding, substrate binding, and catalysis. Here, we describe the application of Raman crystallography to functional RNA molecules. RNA represents unique opportunities and challenges for Raman crystallography. One issue that confounds studies of RNA is its tendency to adopt multiple non-functional folds. Raman crystallography has the advantage that it isolates a single state of the RNA within the crystal and can evaluate its fold, metal ion binding properties (ligand identity, stoichiometry, and affinity), proton binding properties (identity, stoichiometry, and affinity), and catalytic potential. In particular, base-specific stretches can be identified and then associated with the binding of metal ions and protons. Because measurements are carried out in the hanging drop at ambient, rather than cryo, conditions and because RNA crystals tend to be approximately 70% solvent, RNA dynamics and conformational changes become experimentally accessible. This review focuses on experimental setup and procedures, acquisition and interpretation of Raman data, and determination of physicochemical properties of the RNA. Raman crystallographic and solution biochemical experiments on the HDV RNA enzyme are summarized and found to be in excellent agreement. Remarkably, characterization of the crystalline state has proven to help rather than hinder functional characterization of functional RNA, most likely because the tendency of RNA to fold heterogeneously is limited in a crystalline environment. Future applications of Raman crystallography to RNA are briefly discussed.
Asunto(s)
Cristalografía/métodos , ARN/química , Espectrometría Raman/métodos , Catálisis , Iones , Ligandos , Metales/química , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Viral/química , Solventes/químicaRESUMEN
The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pK(a) shifted >2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335-13342], while in crystal structures, it is well-positioned for proton transfer. However, no evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on the rate and Mg(2+) binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously unobserved hydrated magnesium ion interacts with N7 of the cleavage site G.U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data.
Asunto(s)
Virus de la Hepatitis Delta/enzimología , Magnesio/metabolismo , ARN Catalítico/metabolismo , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Hidrólisis , Modelos Moleculares , ARN Catalítico/química , Espectrometría RamanRESUMEN
Divalent cations play critical structural and functional roles in many RNAs. While the hepatitis delta virus (HDV) ribozyme can undergo self-cleavage in the presence of molar concentrations of monovalent cations, divalent cations such as Mg(2+) are required for efficient catalysis under physiological conditions. Moreover, the cleavage reaction can be inhibited with Co(NH(3))(6)(3+), an analogue of Mg(H(2)O)(6)(2+). Here, the binding of Mg(2+) and Co(NH(3))(6)(3+) to the HDV ribozyme is studied by Raman microscopic analysis of crystals. Raman difference spectra acquired at different metal ion conditions reveal changes in the ribozyme. When Mg(2+) alone is introduced to the ribozyme, inner sphere coordination of Mg(H(2)O)(x)(2+) (x = 5) to nonbridging PO(2)(-) oxygen and changes in base stretches and phosphodiester group conformation are observed. In addition, binding of Mg(2+) induces deprotonation of a cytosine assigned to the general acid C75, consistent with solution studies. When Co(NH(3))(6)(3+) alone is introduced, deprotonation of C75 is again observed, as are distinctive changes in base vibrational ring modes and phosphodiester backbone conformation. In contrast to Mg(2+) binding, Co(NH(3))(6)(3+) binding does not perturb PO(2)(-) group vibrations, consistent with its ability to make only outer sphere contacts. Surprisingly, competitive binding studies reveal that Co(NH(3))(6)(3+) ions displace some inner sphere-coordinated magnesium species, including ions coordinated to PO(2)(-) groups or the N7 of a guanine, likely G1 at the active site. These observations contrast with the tenet that Co(NH(3))(6)(3+) ions displace only outer sphere magnesium ions. Overall, our data support two classes of inner sphere Mg(2+)-PO(2)(-) binding sites: sites that Co(NH(3))(6)(3+) can displace and others it cannot.
Asunto(s)
Cobalto/metabolismo , Virus de la Hepatitis Delta/enzimología , Magnesio/metabolismo , ARN Catalítico/metabolismo , Unión Competitiva , Catálisis , Dominio Catalítico , Cationes Bivalentes/química , Cobalto/antagonistas & inhibidores , Cobalto/química , Cristalización , Magnesio/química , Conformación de Ácido Nucleico , ARN Catalítico/química , Espectrometría RamanRESUMEN
Sindbis virus is an enveloped positive-sense RNA virus in the alphavirus genus. The nucleocapsid core contains the genomic RNA surrounded by 240 copies of a single capsid protein. The capsid protein is multifunctional, and its roles include acting as a protease, controlling the specificity of RNA that is encapsidated into nucleocapsid cores, and interacting with viral glycoproteins to promote the budding of mature virus and the release of the genomic RNA into the newly infected cell. The region comprising amino acids 81 to 113 was previously implicated in two processes, the encapsidation of the viral genomic RNA and the stable accumulation of nucleocapsid cores in the cytoplasm of infected cells. In the present study, specific amino acids within this region responsible for the encapsidation of the genomic RNA have been identified. The region that is responsible for nucleocapsid core accumulation has considerable overlap with the region that controls encapsidation specificity.
Asunto(s)
Proteínas de la Cápside/fisiología , Genoma Viral , Virus Sindbis/fisiología , Ensamble de Virus , Secuencia de Aminoácidos/fisiología , Proteínas de la Cápside/genética , Mutación , Nucleocápside , ARN ViralRESUMEN
A Raman microscope and Raman difference spectroscopy are used to detect the vibrational signature of RNA-bound magnesium hydrate in crystals of hepatitis delta virus (HDV) ribozyme and to follow the effects of magnesium hydrate binding to the nonbridging phosphate oxygens in the phosphodiester backbone. There is a correlation between the Raman intensity of the innersphere magnesium hydrate signature peak, near 322 cm-1, and the intensity of the PO2- symmetric stretch, near 1100 cm-1, perturbed by magnesium binding, demonstrating direct observation of -PO2-...Mg2+(H2O)x innersphere complexes. The complexes may be pentahydrates (x = 5) and tetrahydrates (x = 4). The assignment of the Raman feature near 322 cm-1 to a magnesium hydrate species is confirmed by isotope shifts observed in D2O and H218O that are semiquantitatively reproduced by calculations. The standardized intensity changes in the 1100 cm-1 PO2- feature seen upon magnesium hydrate binding indicates that there are approximately 5 innersphere Mg2+...-O2P contacts per HDV molecule when the crystal is exposed to a solution containing 20 mM magnesium.