RESUMEN
The first RNA category of the Critical Assessment of Techniques for Structure Prediction competition was only made possible because of the scientists who provided experimental structures to challenge the predictors. In this article, these scientists offer a unique and valuable analysis of both the successes and areas for improvement in the predicted models. All 10 RNA-only targets yielded predictions topologically similar to experimentally determined structures. For one target, experimentalists were able to phase their x-ray diffraction data by molecular replacement, showing a potential application of structure predictions for RNA structural biologists. Recommended areas for improvement include: enhancing the accuracy in local interaction predictions and increased consideration of the experimental conditions such as multimerization, structure determination method, and time along folding pathways. The prediction of RNA-protein complexes remains the most significant challenge. Finally, given the intrinsic flexibility of many RNAs, we propose the consideration of ensemble models.
Asunto(s)
Biología Computacional , Proteínas , Conformación Proteica , Proteínas/química , Modelos Moleculares , Biología Computacional/métodos , Difracción de Rayos XRESUMEN
As more sequencing data accumulate and novel puzzling genetic regulations are discovered, the need for accurate automated modeling of RNA structure increases. RNA structure modeling from chemical probing experiments has made tremendous progress, however accurately predicting large RNA structures is still challenging for several reasons: RNA are inherently flexible and often adopt many energetically similar structures, which are not reliably distinguished by the available, incomplete thermodynamic model. Moreover, computationally, the problem is aggravated by the relevance of pseudoknots and non-canonical base pairs, which are hardly predicted efficiently. To identify nucleotides involved in pseudoknots and non-canonical interactions, we scrutinized the SHAPE reactivity of each nucleotide of the 188 nt long lariat-capping ribozyme under multiple conditions. Reactivities analyzed in the light of the X-ray structure were shown to report accurately the nucleotide status. Those that seemed paradoxical were rationalized by the nucleotide behavior along molecular dynamic simulations. We show that valuable information on intricate interactions can be deduced from probing with different reagents, and in the presence or absence of Mg2+. Furthermore, probing at increasing temperature was remarkably efficient at pointing to non-canonical interactions and pseudoknot pairings. The possibilities of following such strategies to inform structure modeling software are discussed.
RESUMEN
If the A-form helix is the major structural motif found in RNA, the loops that cap them constitute the second most important family of motifs. Among those, two are overrepresented, GNRA and UNCG tetraloops. Recent surveys of RNA structures deposited in the PDB show that GNRA and UNCG tetraloops can adopt tertiary folds that are very different from their canonical conformations, characterized by the presence of a U-turn of a Z-turn, respectively. Crystallographic data from both a lariat-capping (LC) ribozyme and a group II intron ribozyme reveal that a given UUCG tetraloop can adopt a distinct fold depending on its structural environment. Specifically, when the crystal packing applies relaxed constraints on the loop, the canonical Z-turn conformation is observed. In contrast, a highly packed environment induces "squashing" of the tetraloop by distorting its sugar-phosphate backbone in a specific way that expels the first and fourth nucleobases out of the loop, and falls in van der Waals distance of the last base pair of the helix, taking the place of the pair formed between the first and fourth residues in Z-turn loops. The biological relevance of our observations is supported by the presence of similarly deformed loops in the highly packed environment of the ribosome and in a complex between a dsRNA and a RNase III. The finding that Z-turn loops change conformation under higher molecular packing suggests that, in addition to their demonstrated role in stabilizing RNA folding, they may contribute to the three-dimensional structure of RNA by mediating tertiary interactions with distal residues.
Asunto(s)
Conformación de Ácido Nucleico , ARN/química , Cristalografía por Rayos X , Intrones , ARN Catalítico/químicaRESUMEN
Tumor necrosis factor alpha (TNF-α) is expressed promptly during inflammatory responses. Efficient TNF-α mRNA splicing is achieved through a 3' UTR element that activates RNA-dependent eIF2α protein kinase (PKR). The TNF-α RNA activator, we show, folds into a pseudoknot conserved from teleost fish to humans, critical for PKR activation and mRNA splicing. The pseudoknot constrains the RNA into two double-helical stacks having parallel axes, permitting facile PKR dimerization and trans-autophosphorylation needed for kinase activation. Mutations show that the PKR activator potently enhances splicing without inhibiting translation. eIF2α phosphorylation represses translation and is essential for coping with cellular stress, yet PKR-enabled TNF mRNA splicing depends strictly on eIF2α phosphorylation. Indeed, eIF2α phosphorylation at Serine51 is necessary and sufficient to achieve highly efficient splicing, extending its role from negative control of translation to positive control of splicing. This mechanism, operational in human peripheral blood mononuclear cells (PBMCs), links stress signaling to protective immunity through TNF mRNA splicing rendered efficient upon eIF2α phosphorylation.
Asunto(s)
Secuencia Conservada , Empalme del ARN , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/genética , eIF-2 Quinasa/metabolismo , Regiones no Traducidas 3' , Animales , Línea Celular , Células Cultivadas , Cricetinae , Células HeLa , Humanos , Fosforilación , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , ARN Mensajero/química , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility.
Asunto(s)
Respuesta al Choque Térmico/fisiología , Intrones , Mixomicetos/metabolismo , ARN Catalítico/biosíntesis , Mixomicetos/genética , ARN Catalítico/genéticaRESUMEN
Transfer RNAs (tRNAs) play a key role in protein synthesis as adaptor molecules between messenger RNA and protein sequences on the ribosome. Their discovery in the early sixties provoked a worldwide infatuation with the study of their architecture and their function in the decoding of genetic information. tRNAs are also emblematic molecules in crystallography: the determination of the first tRNA crystal structures represented a milestone in structural biology and tRNAs were for a long period the sole source of information on RNA folding, architecture, and post-transcriptional modifications. Crystallographic data on tRNAs in complex with aminoacyl-tRNA synthetases (aaRSs) also provided the first insight into protein:RNA interactions. Beyond the translation process and the history of structural investigations on tRNA, this review also illustrates the renewal of tRNA biology with the discovery of a growing number of tRNA partners in the cell, the involvement of tRNAs in a variety of regulatory and metabolic pathways, and emerging applications in biotechnology and synthetic biology.
Asunto(s)
Modelos Moleculares , ARN de Transferencia/química , ARN de Transferencia/ultraestructura , Dispersión del Ángulo Pequeño , Difracción de Rayos X/métodos , Simulación por Computador , Conformación ProteicaRESUMEN
Polyacrylamide gel electrophoresis (PAGE) constitutes a powerful technique for the efficient purification of RNA molecules dedicated to applications that require high purity levels. PAGE allows for the fractionation of RNA obtained from cell extracts, chemical or enzymatic synthesis, or modification experiments. Native or denaturing conditions can be chosen for analytical or preparative-scale separations and the nucleotide resolution can be tuned by changing the percentage and reticulation of the gel material. In this protocol, we focus on the preparation of milligram-scale amounts of ~200 nucleotides (nt) RNA molecules that were used in subsequent crystallization experiments.
Asunto(s)
Electroforesis en Gel de Poliacrilamida/métodos , Biología Molecular/métodos , ARN/química , ARN/aislamiento & purificación , Cristalización , Peso Molecular , Polvos , ARN/análisis , Soluciones , Espectrofotometría Ultravioleta , Temperatura , Urea/químicaRESUMEN
As obligatory intracellular parasites, viruses rely on cellular machines to complete their life cycle, and most importantly they recruit the host ribosomes to translate their mRNA. The Hepatitis C viral mRNA initiates translation by directly binding the 40S ribosomal subunit in such a way that the initiation codon is correctly positioned in the P site of the ribosome. Such a property is likely to be central for many viruses, therefore the description of host-pathogen interaction at the molecular level is instrumental to provide new therapeutic targets. In this study, we monitored the 40S ribosomal subunit and the viral RNA structural rearrangement induced upon the formation of the binary complex. We further took advantage of an IRES viral mutant mRNA deficient for translation to identify the interactions necessary to promote translation. Using a combination of structure probing in solution and molecular modeling we establish a whole atom model which appears to be very similar to the one obtained recently by cryoEM. Our model brings new information on the complex, and most importantly reveals some structural rearrangement within the ribosome. This study suggests that the formation of a 'kissing complex' between the viral RNA and the 18S ribosomal RNA locks the 40S ribosomal subunit in a conformation proficient for translation.
Asunto(s)
Hepacivirus/genética , Sitios Internos de Entrada al Ribosoma/genética , ARN Viral/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Sistema Libre de Células , Codón Iniciador/genética , Microscopía por Crioelectrón , Células HeLa , Hepacivirus/metabolismo , Hepacivirus/fisiología , Interacciones Huésped-Patógeno , Humanos , Sustancias Macromoleculares/metabolismo , Sustancias Macromoleculares/ultraestructura , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Viral/química , ARN Viral/metabolismo , Conejos , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismoRESUMEN
In yeast Saccharomyces cerevisiae, ~3% of the lysine transfer RNA acceptor 1 (tRK1) pool is imported into mitochondria while the second isoacceptor, tRK2, fully remains in the cytosol. The mitochondrial function of tRK1 is suggested to boost mitochondrial translation under stress conditions. Strikingly, yeast tRK1 can also be imported into human mitochondria in vivo, and can thus be potentially used as a vector to address RNAs with therapeutic anti-replicative capacity into mitochondria of sick cells. Better understanding of the targeting mechanism in yeast and human is thus critical. Mitochondrial import of tRK1 in yeast proceeds first through a drastic conformational rearrangement of tRK1 induced by enolase 2, which carries this freight to the mitochondrial pre-lysyl-tRNA synthetase (preMSK). The latter may cross the mitochondrial membranes to reach the matrix where imported tRK1 could be used by the mitochondrial translation apparatus. This work focuses on the characterization of the complex that tRK1 forms with human enolases and their role on the interaction between tRK1 and human pre-lysyl-tRNA synthetase (preKARS2).
Asunto(s)
Lisina-ARNt Ligasa/metabolismo , Mitocondrias/metabolismo , Fosfopiruvato Hidratasa/metabolismo , ARN de Transferencia/metabolismo , Saccharomyces cerevisiae/metabolismo , Algoritmos , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Proteínas de Transporte de Catión/metabolismo , Citosol/metabolismo , Bases de Datos de Proteínas , Células Hep G2 , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
This paper is a report of a second round of RNA-Puzzles, a collective and blind experiment in three-dimensional (3D) RNA structure prediction. Three puzzles, Puzzles 5, 6, and 10, represented sequences of three large RNA structures with limited or no homology with previously solved RNA molecules. A lariat-capping ribozyme, as well as riboswitches complexed to adenosylcobalamin and tRNA, were predicted by seven groups using RNAComposer, ModeRNA/SimRNA, Vfold, Rosetta, DMD, MC-Fold, 3dRNA, and AMBER refinement. Some groups derived models using data from state-of-the-art chemical-mapping methods (SHAPE, DMS, CMCT, and mutate-and-map). The comparisons between the predictions and the three subsequently released crystallographic structures, solved at diffraction resolutions of 2.5-3.2 Å, were carried out automatically using various sets of quality indicators. The comparisons clearly demonstrate the state of present-day de novo prediction abilities as well as the limitations of these state-of-the-art methods. All of the best prediction models have similar topologies to the native structures, which suggests that computational methods for RNA structure prediction can already provide useful structural information for biological problems. However, the prediction accuracy for non-Watson-Crick interactions, key to proper folding of RNAs, is low and some predicted models had high Clash Scores. These two difficulties point to some of the continuing bottlenecks in RNA structure prediction. All submitted models are available for download at http://ahsoka.u-strasbg.fr/rnapuzzles/.
Asunto(s)
Biología Computacional/métodos , ARN/química , Cristalografía por Rayos X , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN de Transferencia/química , Programas InformáticosRESUMEN
BACKGROUND: Twin-ribozyme introns represent a complex class of mobile group I introns that harbour a lariat capping (LC) ribozyme and a homing endonuclease gene embedded in a conventional self-splicing group I ribozyme (GIR2). Twin-ribozyme introns have so far been confined to nucleolar DNA in Naegleria amoeboflagellates and the myxomycete Didymium iridis. RESULTS: We characterize structural organization, catalytic properties and molecular evolution of a new twin-ribozyme intron in Allovahlkampfia (Heterolobosea). The intron contains two ribozyme domains with different functions in ribosomal RNA splicing and homing endonuclease mRNA maturation. We found Allovahlkampfia GIR2 to be a typical group IC1 splicing ribozyme responsible for addition of the exogenous guanosine cofactor (exoG), exon ligation and circularization of intron RNA. The Allovahlkampfia LC ribozyme, by contrast, represents an efficient self-cleaving ribozyme that generates a small 2',5' lariat cap at the 5' end of the homing endonuclease mRNA, and thus contributes to intron mobility. CONCLUSIONS: The discovery of a twin-ribozyme intron in a member of Heterolobosea expands the distribution pattern of LC ribozymes. We identify a putative regulatory RNA element (AP2.1) in the Allovahlkampfia LC ribozyme that involves homing endonuclease mRNA coding sequences as an important structural component.
RESUMEN
The lariat-capping (LC) ribozyme is a natural ribozyme isolated from eukaryotic microorganisms. Despite apparent structural similarity to group I introns, the LC ribozyme catalyzes cleavage by a 2',5' branching reaction, leaving the 3' product with a 3-nt lariat cap that functionally substitutes for a conventional mRNA cap in the downstream pre-mRNA encoding a homing endonuclease. We describe the crystal structures of the precleavage and postcleavage LC ribozymes, which suggest that structural features inherited from group I ribozymes have undergone speciation due to profound changes in molecular selection pressure, ultimately giving rise to an original branching ribozyme family. The structures elucidate the role of key elements that regulate the activity of the LC ribozyme by conformational switching and suggest a mechanism by which the signal for branching is transmitted to the catalytic core. The structures also show how conserved interactions twist residues, forming the lariat to join chemical groups involved in branching.
Asunto(s)
Evolución Molecular , Intrones/genética , Modelos Moleculares , ARN Catalítico/química , Transducción de Señal/genética , Cristalografía , Conformación Proteica , Dispersión del Ángulo Pequeño , Selección Genética , Difracción de Rayos XRESUMEN
RNA-mediated biological processes usually require precise definition of 5' and 3' ends. RNA ends obtained by in vitro transcription using T7 RNA polymerase are often heterogeneous in length and sequence. An efficient strategy to overcome these drawbacks consists of inserting an RNA with known boundaries in between two ribozymes, usually a 5' hammerhead and a 3' hepatitis delta virus ribozymes, that cleave off the desired RNA. In practice, folding of the three RNAs challenges each other, potentially preventing thorough processing. Folding and cleavage of the 5' hammerhead ribozyme relies on a sequence of nucleotides belonging to the central RNA making it more sensitive than the usual 3' hepatitis delta virus ribozyme. The intrinsic stability of the central RNA may thus prevent correct processing of the full transcript. Here, we present a method in which incorporation of a full-length hammerhead ribozyme with a specific tertiary interaction prevents alternative folding with the lariat capping GIR1 ribozyme and enables complete cleavage in the course of the transcription. This strategy may be transposable for in vitro transcription of any highly structured RNA.
Asunto(s)
ARN Catalítico/metabolismo , ARN de Helminto/metabolismo , ARN/metabolismo , Schistosoma/enzimología , Animales , Secuencia de Bases , Northern Blotting/métodos , Clonación Molecular/métodos , Simulación por Computador , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/química , ARN/genética , ARN Catalítico/química , ARN Catalítico/genética , ARN de Helminto/química , ARN de Helminto/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Schistosoma/química , Schistosoma/genética , Schistosoma/metabolismo , Programas Informáticos , Transcripción GenéticaRESUMEN
Ribonuclease P RNA requires a sharply kinked RNA helix to make a loop-receptor interaction that creates the binding site for the substrate. In some forms of the ribozyme, this is accomplished by a k-turn, while others have a different element called the pk-turn. The structure of the pk-turn in RNase P of Thermotoga maritima is globally very similar to a k-turn, but lacks all the standard features of that structure, including long-range hydrogen bonds between the two helical arms. We show here that in an isolated RNA duplex, the pk-turn fails to adopt a tightly kinked structure, but rather is a flexible element. This suggests that the tertiary contacts of RNase P assist its folding into the required kinked structure. We find that we can replace the k-turn of the SAM-I riboswitch with the pk-turn, such that the resulting RNA retains its ability to bind SAM, although with lower affinity. We also find that we can replace the pk-turn of T. maritima RNase P with a standard k-turn (in either orientation) with retention of ribozyme activity. Thus, although the pk-turn cannot intrinsically fold into the kinked structure, it can be induced to fold correctly in context. And the pk-turn and k-turns can substitute functionally for one another.
Asunto(s)
ARN Bacteriano/química , Ribonucleasa P/química , Thermotoga maritima/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transferencia Resonante de Energía de Fluorescencia , Enlace de Hidrógeno , Modelos Moleculares , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Pliegue del ARN , Estabilidad del ARN , Ribonucleasa P/genética , Riboswitch , Thermotoga maritima/químicaRESUMEN
The endoribonuclease III (RNase III) belongs to the enzyme family known to process double-stranded RNAs. Staphylococcus aureus RNase III was shown to regulate, in concert with the quorum sensing induced RNAIII, the degradation of several mRNAs encoding virulence factors and the transcriptional repressor of toxins Rot. Two of the mRNA-RNAIII complexes involve fully base paired loop-loop interactions with similar sequences that are cleaved by RNase III at a unique position. We show here that the sequence of the base pairs within the loop-loop interaction is not critical for RNase III cleavage, but that the co-axial stacking of three consecutive helices provides an ideal topology for RNase III recognition. In contrast, RNase III induces several strong cleavages in a regular helix, which carries a sequence similar to the loop-loop interaction. The introduction of a bulged loop that interrupts the regular helix restrains the number of cleavages. This work shows that S. aureus RNase III is able to bind and cleave a variety of RNA-mRNA substrates, and that specific structure elements direct the action of RNase III.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/metabolismo , Ribonucleasa III/metabolismo , Staphylococcus aureus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Activación Enzimática , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Porinas/genética , Porinas/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Mapeo de Interacción de Proteínas , Percepción de Quorum , Estabilidad del ARN , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasa III/genética , Staphylococcus aureus/genética , Relación Estructura-ActividadRESUMEN
RNA structures are built from recurrent modules that can be identified by structural and comparative sequence analysis. In order to assemble sets of helices in compact architectures, modules that introduce bends and kinks are necessary. Among such modules, kink-turns form an important family that presents sequence and structural characteristics. Here, we describe an internal loop in the bacterial type A RNase P RNA that sets helices bound at the junctions exactly in the same relative positions as in kink-turns but without the structural signatures typical of kink-turns. Our work suggests that identifying a structural module in a subset of RNA sequences constitutes a strategy to identify distinct sequential motifs sharing common structural characteristics.
Asunto(s)
Modelos Moleculares , Ribonucleasa P/química , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Motivos de Nucleótidos , ARN Bacteriano/química , Alineación de SecuenciaRESUMEN
Apart from the ribosome, the crystal structure of the bacterial RNase P in complex with a tRNA, reported by Reiter and colleagues recently, constitutes the first example of a multiple turnover RNA enzyme. Except in rare exceptions, RNase P is ubiquitous and, like the ribosome, is older than the initial branch point of the phylogenetic tree. Importantly, the structure shows how the RNA and the protein moieties cooperate to process the pre-tRNA substrates. The catalytic site comprises some critical RNA residues spread over the secondary structure but gathered in a compact volume next to the protein, which helps recognize and orient the substrate. The discussion here outlines some important aspects of that crystal structure, some of which could apply to RNA molecules in general.
Asunto(s)
Bacillus subtilis/genética , Escherichia coli/genética , ARN Bacteriano/genética , Ribonucleasa P/química , Thermotoga maritima/genética , Secuencia de Aminoácidos , Bacillus subtilis/enzimología , Emparejamiento Base , Dominio Catalítico , Cristalografía , Escherichia coli/enzimología , Evolución Molecular , Holoenzimas , Datos de Secuencia Molecular , Filogenia , Estructura Cuaternaria de Proteína , Precursores del ARN/metabolismo , ARN Bacteriano/química , ARN Bacteriano/metabolismo , ARN Catalítico/química , ARN Catalítico/genética , ARN Catalítico/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Ribonucleasa P/genética , Ribonucleasa P/metabolismo , Especificidad por Sustrato , Thermotoga maritima/enzimologíaRESUMEN
RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes.
Asunto(s)
Endonucleasas/genética , ARN Catalítico/química , ARN Catalítico/metabolismo , ARN Mensajero/metabolismo , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , ARN Catalítico/genética , ARN Mensajero/química , ARN Mensajero/genéticaRESUMEN
A general approach for modeling the architecture of large and structured RNA molecules is described. The method exploits the modularity and the hierarchical folding of RNA architecture that is viewed as the assembly of preformed double-stranded helices defined by Watson-Crick base pairs and RNA modules maintained by non-Watson-Crick base pairs. Despite the extensive molecular neutrality observed in RNA structures, specificity in RNA folding is achieved through global constraints like lengths of helices, coaxiality of helical stacks, and structures adopted at the junctions of helices. The Assemble integrated suite of computer tools allows for sequence and structure analysis as well as interactive modeling by homology or ab initio assembly with possibilities for fitting within electronic density maps. The local key role of non-Watson-Crick pairs guides RNA architecture formation and offers metrics for assessing the accuracy of three-dimensional models in a more useful way than usual root mean square deviation (RMSD) values.
Asunto(s)
Modelos Moleculares , Conformación de Ácido Nucleico , Proteínas de Unión al ARN/metabolismo , ARN/química , Secuencias Reguladoras de Ácido Ribonucleico/genética , Programas Informáticos , Secuencia de Bases , ARN/metabolismo , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
In vitro transcription is a simple procedure that allows for template-directed synthesis of RNA molecules of any sequence from short oligonucleotides to those of several kilobases in µg to mg quantities. It is based on the engineering of a template that includes a bacteriophage promoter sequence (e.g. from the T7 coliphage) upstream of the sequence of interest followed by transcription using the corresponding RNA polymerase. In vitro transcripts are used in analytical techniques (e.g. hybridization analysis), structural studies (for NMR and X-ray crystallography), in biochemical and genetic studies (e.g. as antisense reagents), and as functional molecules (ribozymes and aptamers).