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.     

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.     

Cleavage of pre-tRNAs by the splicing endonuclease requires a composite active site

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5' and 3' splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively. A recent structure of an archaeal endonuclease complexed with a bulge-helix-bulge RNA has led to the unexpected hypothesis that catalysis requires a critical 'cation-pi sandwich' composed of two arginine residues that serve to position the RNA substrate within the active site. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5' splice site requires the conserved cation-pi sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.     

Unravelling the dynamics of RNA degradation by ribonuclease II and its RNA-bound complex

RNA degradation is a determining factor in the control of gene expression. The maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. Ribonuclease II (RNase II) is a major exoribonuclease that intervenes in all of these fundamental processes; it can act independently or as a component of the exosome, an essential RNA-degrading multiprotein complex. RNase II-like enzymes are found in all three kingdoms of life, but there are no structural data for any of the proteins of this family. Here we report the X-ray crystallographic structures of both the ligand-free (at 2.44 A resolution) and RNA-bound (at 2.74 A resolution) forms of Escherichia coli RNase II. In contrast to sequence predictions, the structures show that RNase II is organized into four domains: two cold-shock domains, one RNB catalytic domain, which has an unprecedented alphabeta-fold, and one S1 domain. The enzyme establishes contacts with RNA in two distinct regions, the 'anchor' and the 'catalytic' regions, which act synergistically to provide catalysis. The active site is buried within the RNB catalytic domain, in a pocket formed by four conserved sequence motifs. The structure shows that the catalytic pocket is only accessible to single-stranded RNA, and explains the specificity for RNA versus DNA cleavage. It also explains the dynamic mechanism of RNA degradation by providing the structural basis for RNA translocation and enzyme processivity. We propose a reaction mechanism for exonucleolytic RNA degradation involving key conserved residues. Our three-dimensional model corroborates all existing biochemical data for RNase II, and elucidates the general basis for RNA degradation. Moreover, it reveals important structural features that can be extrapolated to other members of this family.     

Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5' end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5' end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5' phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, provide direct support for the 5' region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5' end of the guide RNA.     

Crystal structure of a hairpin ribozyme-inhibitor complex with implications for catalysis

The hairpin ribozyme catalyses sequence-specific cleavage of RNA. The active site of this natural RNA results from the docking of two irregular helices: stems A and B. One strand of stem A harbours the scissile bond. The 2.4 A resolution structure of a hairpin ribozyme-inhibitor complex reveals that the ribozyme aligns the 2'-OH nucleophile and the 5'-oxo leaving group by twisting apart the nucleotides that flank the scissile phosphate. The base of the nucleotide preceding the cleavage site is stacked within stem A; the next nucleotide, a conserved guanine, is extruded from stem A and accommodated by a highly complementary pocket in the minor groove of stem B. Metal ions are absent from the active site. The bases of four conserved purines are positioned potentially to serve as acid-base catalysts. This is the first structure determination of a fully assembled ribozyme active site that catalyses a phosphodiester cleavage without recourse to metal ions.     

Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex

RNA interference and related RNA silencing phenomena use short antisense guide RNA molecules to repress the expression of target genes. Argonaute proteins, containing amino-terminal PAZ (for PIWI/Argonaute/Zwille) domains and carboxy-terminal PIWI domains, are core components of these mechanisms. Here we show the crystal structure of a Piwi protein from Archaeoglobus fulgidus (AfPiwi) in complex with a small interfering RNA (siRNA)-like duplex, which mimics the 5' end of a guide RNA strand bound to an overhanging target messenger RNA. The structure contains a highly conserved metal-binding site that anchors the 5' nucleotide of the guide RNA. The first base pair of the duplex is unwound, separating the 5' nucleotide of the guide from the complementary nucleotide on the target strand, which exits with the 3' overhang through a short channel. The remaining base-paired nucleotides assume an A-form helix, accommodated within a channel in the PIWI domain, which can be extended to place the scissile phosphate of the target strand adjacent to the putative slicer catalytic site. This study provides insights into mechanisms of target mRNA recognition and cleavage by an Argonaute-siRNA guide complex.     

Structure of yeast Argonaute with guide RNA

The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 ? crystal structure of Kluyveromyces polysporus Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2'-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation analyses and analogies to ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for RNA cleavage.     

Structural basis for translation termination on the 70S ribosome

At termination of protein synthesis, type I release factors promote hydrolysis of the peptidyl-transfer RNA linkage in response to recognition of a stop codon. Here we describe the crystal structure of the Thermus thermophilus 70S ribosome in complex with the release factor RF1, tRNA and a messenger RNA containing a UAA stop codon, at 3.2 A resolution. The stop codon is recognized in a pocket formed by conserved elements of RF1, including its PxT recognition motif, and 16S ribosomal RNA. The codon and the 30S subunit A site undergo an induced fit that results in stabilization of a conformation of RF1 that promotes its interaction with the peptidyl transferase centre. Unexpectedly, the main-chain amide group of Gln 230 in the universally conserved GGQ motif of the factor is positioned to contribute directly to peptidyl-tRNA hydrolysis.     

Structure of a tyrosyl-tRNA synthetase splicing factor bound to a group I intron RNA

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.     

Crystal structure of a self-splicing group I intron with both exons

The discovery of the RNA self-splicing group I intron provided the first demonstration that not all enzymes are proteins. Here we report the X-ray crystal structure (3.1-A resolution) of a complete group I bacterial intron in complex with both the 5'- and the 3'-exons. This complex corresponds to the splicing intermediate before the exon ligation step. It reveals how the intron uses structurally unprecedented RNA motifs to select the 5'- and 3'-splice sites. The 5'-exon's 3'-OH is positioned for inline nucleophilic attack on the conformationally constrained scissile phosphate at the intron-3'-exon junction. Six phosphates from three disparate RNA strands converge to coordinate two metal ions that are asymmetrically positioned on opposing sides of the reactive phosphate. This structure represents the first splicing complex to include a complete intron, both exons and an organized active site occupied with metal ions.     

Snapshots of tRNA sulphuration via an adenylated intermediate

Uridine at the first anticodon position (U34) of glutamate, lysine and glutamine transfer RNAs is universally modified by thiouridylase into 2-thiouridine (s2U34), which is crucial for precise translation by restricting codon-anticodon wobble during protein synthesis on the ribosome. However, it remains unclear how the enzyme incorporates reactive sulphur into the correct position of the uridine base. Here we present the crystal structures of the MnmA thiouridylase-tRNA complex in three discrete forms, which provide snapshots of the sequential chemical reactions during RNA sulphuration. On enzyme activation, an alpha-helix overhanging the active site is restructured into an idiosyncratic beta-hairpin-containing loop, which packs the flipped-out U34 deeply into the catalytic pocket and triggers the activation of the catalytic cysteine residues. The adenylated RNA intermediate is trapped. Thus, the active closed-conformation of the complex ensures accurate sulphur incorporation into the activated uridine carbon by forming a catalytic chamber to prevent solvent from accessing the catalytic site. The structures of the complex with glutamate tRNA further reveal how MnmA specifically recognizes its three different tRNA substrates. These findings provide the structural basis for a general mechanism whereby an enzyme incorporates a reactive atom at a precise position in a biological molecule.     

Mechanism of transfer RNA maturation by CCA-adding enzyme without using an oligonucleotide template

Transfer RNA nucleotidyltransferases (CCA-adding enzymes) are responsible for the maturation or repair of the functional 3' end of tRNAs by means of the addition of the essential nucleotides CCA. However, it is unclear how tRNA nucleotidyltransferases polymerize CCA onto the 3' terminus of immature tRNAs without using a nucleic acid template. Here we describe the crystal structure of the Archaeoglobus fulgidus tRNA nucleotidyltransferase in complex with tRNA. We also present ternary complexes of this enzyme with both RNA duplex mimics of the tRNA acceptor stem that terminate with the nucleotides C74 or C75, as well as the appropriate incoming nucleoside 5'-triphosphates. A single nucleotide-binding pocket exists whose specificity for both CTP and ATP is determined by the protein side chain of Arg 224 and backbone phosphates of the tRNA, which are non-complementary to and thus exclude UTP and GTP. Discrimination between CTP or ATP at a given addition step and at termination arises from changes in the size and shape of the nucleotide binding site that is progressively altered by the elongating 3' end of the tRNA.     

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.     

Control of RNase E-mediated RNA degradation by 5'-terminal base pairing in E. coli.

Despite the variety of messenger RNA half-lives in bacteria (0.5-30 min in Escherichia coli) and their importance in controlling gene expression, their molecular basis remains obscure. The lifetime of an entire mRNA molecule can be determined by features near its 5' end, but no 5' exoribonuclease has been identified in any prokaryotic organism. A mutation that inactivates E. coli RNase E also increases the average lifetime of bulk E. coli mRNA and of many individual messages, suggesting that cleavage by this endonuclease may be the rate-determining step in the degradation of most mRNAs in E. coli. We have investigated the substrate preference of RNase E in E. coli by using variants of RNA I, a small untranslated RNA whose swift degradation in vivo is initiated by RNase E cleavage at an internal site. We report here that RNase E has an unprecedented substrate specificity for an endoribonuclease, as it preferentially cleaves RNAs that have several unpaired nucleotides at the 5' end. The sensitivity of RNase E to 5'-terminal base pairing may explain how determinants near the 5' end can control rates of mRNA decay in bacteria.     

An aminoacyl tRNA synthetase whose sequence fits into neither of the two known classes

Aminoacyl transfer RNA synthetases catalyse the first step of protein synthesis and establish the rules of the genetic code through the aminoacylation of tRNAs. There is a distinct synthetase for each of the 20 amino acids and throughout evolution these enzymes have been divided into two classes of ten enzymes each. These classes are defined by the distinct architectures of their active sites, which are associated with specific and universal sequence motifs. Because the synthesis of aminoacyl-tRNAs containing each of the twenty amino acids is a universally conserved, essential reaction, the absence of a recognizable gene for cysteinyl tRNA synthetase in the genomes of Archae such as Methanococcus jannaschii and Methanobacterium thermoautotrophicum has been difficult to interpret. Here we describe a different cysteinyl-tRNA synthetase from M. jannaschii and Deinococcus radiodurans and its characterization in vitro and in vivo. This protein lacks the characteristic sequence motifs seen in the more than 700 known members of the two canonical classes of tRNA synthetase and may be of ancient origin. The existence of this protein contrasts with proposals that aminoacylation with cysteine in M. jannaschii is an auxiliary function of a canonical prolyl-tRNA synthetase.