A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA

Hepatitis delta virus genomic and antigenomic RNAs contain a self-cleavage site hypothesized to function in processing the viral RNA during replication. Self-cleavage requires only a divalent cation and is mediated at the genomic site by a sequence of less than 85 nucleotides. We propose that the genomic self-cleaving sequence element and a corresponding sequence from the anti-genomic RNA could generate related secondary structures. The region of the antigenomic sequence, predicted from the proposed structure, was synthesized and shown to be sufficient for self-cleavage. Evidence for two stems which form a tertiary interaction was obtained by site-specific mutagenesis of the antigenomic sequence. Efficient self-cleavage in 10 M formamide or 5 M urea, also a property of the genomic sequence, was dependent on base-pairing in both stems. But in the absence of denaturants, the stem distal to the site of cleavage was not required, suggesting that the tertiary interaction stabilizes the structure required for self-cleavage.     

Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups.

Shortened forms of the group I intron from Tetrahymena catalyse sequence-specific cleavage of exogenous oligonucleotide substrates. The association between RNA enzyme (ribozyme) and substrate is mediated by pairing between an internal guide sequence on the ribozyme and a complementary sequence on the substrate. RNA substrates and cleavage products associate with a binding energy greater than that of base-pairing by approximately 4 kcal-mol-1 (at 42 degrees C), whereas DNA associates with an energy around that expected for base-pairing. It has been proposed that the difference in binding affinity is due to specific 2'-OH groups on an RNA substrate forming stabilizing tertiary interactions with the core of the ribozyme, or that the RNA.RNA helix formed upon association of an RNA substrate and the ribozyme might be more stable than an RNA.DNA helix of the same sequence. To differentiate between these two models, chimaeric oligonucleotides containing deoxynucleotide residues at successive positions along the chain were synthesized, and their equilibrium binding constants for association with the ribozyme were measured directly by a new gel electrophoresis technique. We report here that most of the extra binding energy can be accounted for by discrete RNA-ribozyme interactions, the 2'-OH group on the sugar residue three nucleotides from the cleavage site contributing the most interaction energy. Thus, in addition to the well documented binding of RNA to RNA by base-pairing, 2'-OH groups within a duplex can also mediate association between RNA molecules.     

Phylogenetic and genetic evidence for base-triples in the catalytic domain of group I introns

Understanding the mechanisms by which ribozymes catalyse chemical reactions requires a detailed knowledge of their structure. The secondary structure of the group I introns has been confirmed by comparison of over 70 published sequences, by chemical protection studies, and by genetic experiments involving compensatory mutations. Phylogenetic data can also be used to identify tertiary interactions in RNA molecules. This was first done by Levitt, who predicted tertiary interactions in transfer RNA, which were subsequently confirmed by X-ray crystallography. More recently, sequence comparison data have been used to predict tertiary interactions in ribosomal RNA. We have searched a complete alignment of the core regions of group I introns for evolutionary covariations that could not be ascribed to classical Watson-Crick or wobble base pairings. Here we describe two examples of phylogenetic covariation that are most simply explained by postulating hydrogen-bonded base-triples similar to those found in tRNA. Genetic experiments with the Tetrahymena and sunY introns confirm the importance of these interactions for the structure of the ribozyme.     

Structure of the Escherichia coli ribosomal termination complex with release factor 2

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.     

Crystal structure of the RNA component of bacterial ribonuclease P

Transfer RNA (tRNA) is produced as a precursor molecule that needs to be processed at its 3' and 5' ends. Ribonuclease P is the sole endonuclease responsible for processing the 5' end of tRNA by cleaving the precursor and leading to tRNA maturation. It was one of the first catalytic RNA molecules identified and consists of a single RNA component in all organisms and only one protein component in bacteria. It is a true multi-turnover ribozyme and one of only two ribozymes (the other being the ribosome) that are conserved in all kingdoms of life. Here we show the crystal structure at 3.85 A resolution of the RNA component of Thermotoga maritima ribonuclease P. The entire RNA catalytic component is revealed, as well as the arrangement of the two structural domains. The structure shows the general architecture of the RNA molecule, the inter- and intra-domain interactions, the location of the universally conserved regions, the regions involved in pre-tRNA recognition and the location of the active site. A model with bound tRNA is in agreement with all existing data and suggests the general basis for RNA-RNA recognition by this ribozyme.     

A tertiary interaction that links active-site domains to the 5' splice site of a group II intron

Group II introns are self-splicing RNAs that are commonly found in the genes of plants, fungi, yeast and bacteria. Little is known about the tertiary structure of group II introns, which are among the largest natural ribozymes. The most conserved region of the intron is domain 5 (D5), which, together with domain 1 (D1), is required for all reactions catalysed by the intron. Despite the importance of D5, its spatial relationship and tertiary contacts to other active-site constituents have remained obscure. Furthermore, D5 has never been placed directly at a site of catalysis by the intron. Here we show that a set of tertiary interactions (lambda-lambda') links catalytically essential regions of D5 and D1, creating the framework for an active-site and anchoring it at the 5' splice site. Highly conserved elements similar to components of the lambda-lambda' interaction are found in the eukaryotic spliceosome.     

A 3' splice site-binding sequence in the catalytic core of a group I intron

Ribozymes use specific RNA-RNA interactions for substrate binding and active-site formation. Self-splicing group I introns have approximately 70 nucleotides constituting the core, a region containing sequences and structures indispensable for catalytic function. The catalytic core must interact with the substrates used for the two steps of the self-splicing reaction, that is, guanosine, the 5'-splice-site helix (P1) and the 3' splice site. Mutational evidence suggests that core sequences near segment J6/7 that joins the base-paired stems P6 and P7, and the bulged base of P7(5'), participate in binding guanosine substrate, but nothing is known about the interactions between the core, the 5'-splice-site helix and the 3' splice site. On the basis of comparative sequence data, it has been suggested that two specific bases in the catalytic core of group I introns might form a binding sequence for the 3' splice site. Here we present genetic evidence that such a binding site exists in the core of the Tetrahymena large subunit ribosomal RNA intron. We demonstrate that this pairing, termed P9.0, is functionally important in the exon ligation step of self-splicing, but is not itself responsible for 3'-splice-site selection.     

Mechanism of recognition of the 5' splice site in self-splicing group I introns

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.     

Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA

Ribonuclease (RNase) P is the universal ribozyme responsible for 5'-end tRNA processing. We report the crystal structure of the Thermotoga maritima RNase P holoenzyme in complex with tRNA(Phe). The 154?kDa complex consists of a large catalytic RNA (P RNA), a small protein cofactor and a mature tRNA. The structure shows that RNA-RNA recognition occurs through shape complementarity, specific intermolecular contacts and base-pairing interactions. Soaks with a pre-tRNA 5' leader sequence with and without metal help to identify the 5' substrate path and potential catalytic metal ions. The protein binds on top of a universally conserved structural module in P RNA and interacts with the leader, but not with the mature tRNA. The active site is composed of phosphate backbone moieties, a universally conserved uridine nucleobase, and at least two catalytically important metal ions. The active site structure and conserved RNase P-tRNA contacts suggest a universal mechanism of catalysis by RNase P.     

Structure of Sindbis virus core protein reveals a chymotrypsin-like serine proteinase and the organization of the virion

Sindbis virus consists of a nucleocapsid core surrounded by a lipid membrane through which penetrate 80 glycoprotein trimers. The structure of the core protein comprising the coat surrounding the genomic RNA has been determined. The polypeptide fold from residue 114 to residue 264 is homologous to that of chymotrypsin-like serine proteinases with catalytic residues His 141, Asp 163 and Ser 215 of the core protein positioned as in other serine proteinases. The C-terminal tryptophan remains in the P1 substrate site subsequent to the autocatalytic cis cleavage of the capsid protein, thus rendering the proteinase inactive. Model building of the Sindbis core protein dimer shows that the nucleocapsid is likely to have T = 4 quasisymmetry.     

lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements

Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs whose 3'-untranslated regions (3' UTRs) bind to STAU1, a protein that binds to double-stranded RNA. Earlier studies defined the STAU1-binding site within ADP-ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex. However, we were unable to identify comparable structures in the 3' UTRs of other targets of SMD. Here we show that STAU1-binding sites can be formed by imperfect base-pairing between an Alu element in the 3' UTR of an SMD target and another Alu element in a cytoplasmic, polyadenylated long non-coding RNA (lncRNA). An individual lncRNA can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions of non-coding RNAs and Alu elements. Not all mRNAs that contain an Alu element in the 3' UTR are targeted for SMD even in the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides, and these include small nucleolar RNAs and microRNAs. Our finding that the binding of STAU1 to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNAs. We name these lncRNAs half-STAU1-binding site RNAs (1/2-sbsRNAs).     

人SEPT9蛋白同源建模和活性位点分析

利用生物信息学在线软件预测了人SETP 9蛋白质的二级结构和模体信息,同时对其三级结构进行同源建模和模建结果质量评价,其次预测了该蛋白质的活性位点信息,旨在从蛋白质序列特征和分子结构水平理解其在人类生理病理过程中的作用.结果表明,模建的SEPT 9蛋白结构品质较高,具有7段α-螺旋和2组β-折叠结构,是一个典型的α/β类蛋白,表面呈弱正电势分布;人SEPT 9蛋白具有8个不同模体,可能参与不同生化反应或执行不同的功能.搜寻获得了人SEPT 9蛋白配基结合位点有10个,其中位点1可能是该蛋白的活性位点.这些研究结果对理解人SEPT 9蛋白功能以及配基结合位点定位非常重要,也为针对SEPT 9蛋白的分子对接和药物从头设计提供了理论基础.     

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin （HA） cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondary structure with RNAstructure 4.1 program in an iterative extension process. Statistical analysis of the sequences showed that the HA cleavage site basic amino acids favor the adenine-rich codons, and the corresponding mRNA fragments are mainly in the folding states of single-stranded loops. Our sequential and structural analyses showed that to prevent and control these highly pathogenic viruses, that is, to inhibit the gene expression of avian influenza virus H5N1 subtypes, we should consider the single-stranded loop regions of the HA cleavage site-coding sequences as the targets of RNA interference.     

Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA.

The discovery of RNA enzymes has, for the first time, provided a single molecule that has both genetic and catalytic properties. We have devised techniques for the mutation, selection and amplification of catalytic RNA, all of which can be performed rapidly in vitro. Here we describe how these techniques can be integrated and performed repeatedly within a single reaction vessel. This allows evolution experiments to be carried out in response to artificially imposed selection constraints. We worked with the Tetrahymena ribozyme, a self-splicing group I intron derived from the large ribosomal RNA precursor of Tetrahymena thermophila that catalyses sequence-specific phosphoester transfer reactions involving RNA substrates. It consists of 413 nucleotides, and assumes a well-defined secondary and tertiary structure responsible for its catalytic activity. We selected for variant forms of the enzyme that could best react with a DNA substrate. This led to the recovery of a mutant form of the enzyme that cleaves DNA more efficiently than the wild-type enzyme. The selected molecule represents the discovery of the first RNA enzyme known to cleave single-stranded DNA specifically.     

Crystal structure of the specificity domain of ribonuclease P

RNase P is the only endonuclease responsible for processing the 5' end of transfer RNA by cleaving a precursor and leading to tRNA maturation. It contains an RNA component and a protein component and has been identified in all organisms. It was one of the first catalytic RNAs identified and the first that acts as a multiple-turnover enzyme in vivo. RNase P and the ribosome are so far the only two ribozymes known to be conserved in all kingdoms of life. The RNA component of bacterial RNase P can catalyse pre-tRNA cleavage in the absence of the RNase P protein in vitro and consists of two domains: a specificity domain and a catalytic domain. Here we report a 3.15-A resolution crystal structure of the 154-nucleotide specificity domain of Bacillus subtilis RNase P. The structure reveals the architecture of this domain, the interactions that maintain the overall fold of the molecule, a large non-helical but well-structured module that is conserved in all RNase P RNA, and the regions that are involved in interactions with the substrate.