Recognition of small interfering RNA by a viral suppressor of RNA silencing

RNA silencing (also known as RNA interference) is a conserved biological response to double-stranded RNA that regulates gene expression, and has evolved in plants as a defence against viruses. The response is mediated by small interfering RNAs (siRNAs), which guide the sequence-specific degradation of cognate messenger RNAs. As a counter-defence, many viruses encode proteins that specifically inhibit the silencing machinery. The p19 protein from the tombusvirus is such a viral suppressor of RNA silencing and has been shown to bind specifically to siRNA. Here, we report the 1.85-A crystal structure of p19 bound to a 21-nucleotide siRNA, where the 19-base-pair RNA duplex is cradled within the concave face of a continuous eight-stranded beta-sheet, formed across the p19 homodimer interface. Direct and water-mediated intermolecular contacts are restricted to the backbone phosphates and sugar 2'-OH groups, consistent with sequence-independent p19-siRNA recognition. Two alpha-helical 'reading heads' project from opposite ends of the p19 homodimer and position pairs of tryptophans for stacking over the terminal base pairs, thereby measuring and bracketing both ends of the siRNA duplex. Our structure provides an illustration of siRNA sequestering by a viral protein.     

Structural basis for site-specific ribose methylation by box C/D RNA protein complexes

Box C/D RNA protein complexes (RNPs) direct site-specific 2'-O-methylation of RNA and ribosome assembly. The guide RNA in C/D RNP forms base pairs with complementary substrates and selects the modification site using a molecular ruler. Despite many studies of C/D RNP structure, the fundamental questions of how C/D RNAs assemble into RNPs and how they guide modification remain unresolved. Here we report the crystal structure of an entire catalytically active archaeal C/D RNP consisting of a bipartite C/D RNA associated with two substrates and two copies each of Nop5, L7Ae and fibrillarin at 3.15-? resolution. The substrate pairs with the second through the eleventh nucleotide of the 12-nucleotide guide, and the resultant duplex is bracketed in a channel with flexible ends. The methyltransferase fibrillarin binds to an undistorted A-form structure of the guide-substrate duplex and specifically loads the target ribose into the active site. Because interaction with the RNA duplex alone does not determine the site specificity, fibrillarin is further positioned by non-specific and specific protein interactions. Compared with the structure of the inactive C/D RNP, extensive domain movements are induced by substrate loading. Our results reveal the organization of a monomeric C/D RNP and the mechanism underlying its site-specific methylation activity.     

Interconversion of single and double helices formed from synthetic molecular strands

Synthetic single-helical conformations are quite common, but the formation of double helices based on recognition between the two constituent strands is relatively rare. Known examples include duplex formation through base-pair-specific hydrogen bonding and stacking, as found in nucleic acids and their analogues, and polypeptides composed of amino acids with alternating L and D configurations. Some synthetic polymers and self-assembled fibres have double-helical winding induced by van der Waals interactions. A third mode of non-covalent interaction, coordination of organic ligands to metal ions, can give rise to double, triple and quadruple helices, although in this case the assembly is driven by the coordination geometry of the metal and the structure of the ligands, rather than by direct inter-strand complementarity. Here we describe a family of oligomeric molecules with bent conformations, which exhibit dynamic exchange between single and double molecular helices in solution, through spiral sliding of the synthetic oligomer strands. The bent conformations leading to the helical shape of the molecules result from intramolecular hydrogen bonding within 2'-pyridyl-2-pyridinecarboxamide units, with extensive intermolecular aromatic stacking stabilizing the double-stranded helices that form through dimerization.     

A ribozyme composed of only two different nucleotides

RNA molecules are thought to have been prominent in the early history of life on Earth because of their ability both to encode genetic information and to exhibit catalytic function. The modern genetic alphabet relies on two sets of complementary base pairs to store genetic information. However, owing to the chemical instability of cytosine, which readily deaminates to uracil, a primitive genetic system composed of the bases A, U, G and C may have been difficult to establish. It has been suggested that the first genetic material instead contained only a single base-pairing unit. Here we show that binary informational macromolecules, containing only two different nucleotide subunits, can act as catalysts. In vitro evolution was used to obtain ligase ribozymes composed of only 2,6-diaminopurine and uracil nucleotides, which catalyse the template-directed joining of two RNA molecules, one bearing a 5'-triphosphate and the other a 3'-hydroxyl. The active conformation of the fastest isolated ribozyme had a catalytic rate that was about 36,000-fold faster than the uncatalysed rate of reaction. This ribozyme is specific for the formation of biologically relevant 3',5'-phosphodiester linkages.     

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.     

The three-dimensional structure of a DNA duplex containing looped-out bases

Unpaired bases in DNA have been assigned a possible role in the mechanism of frameshift mutagenesis in sequences with repeated base pairs. They also occur in quasipalindromic DNA sequences, which have been implicated in mutagenesis where there are no repeated base pairs, through the formation of single-stranded hairpin loops. The conformation of unpaired bases in DNA has been the subject of numerous thermodynamic as well as high resolution NMR (nuclear magnetic resonance) studies (reviewed in ref. 4). The NMR studies in solution have shown that the duplex of the tridecamer DNA fragment d(CGCAGAATTCGCG) remains intact, and that the unpaired adenosines are stacked into the duplex. Having crystallized this oligonucleotide and determined its structure, we find its conformation in the crystal is close to that of a B-DNA duplex, with the two additional adenosines looped out from the double helix and causing little disruption of the rest of the structure.     

Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain

The inhibitor-of-apoptosis proteins (IAPs) regulate programmed cell death by inhibiting members of the caspase family of enzymes. Recently, a mammalian protein called Smac (also named DIABLO) was identified that binds to the IAPs and promotes caspase activation. Although undefined in the X-ray structure, the amino-terminal residues of Smac are critical for its function. To understand the structural basis for molecular recognition between Smac and the IAPs, we determined the solution structure of the BIR3 domain of X-linked IAP (XIAP) complexed with a functionally active nine-residue peptide derived from the N terminus of Smac. The peptide binds across the third beta-strand of the BIR3 domain in an extended conformation with only the first four residues contacting the protein. The complex is stabilized by four intermolecular hydrogen bonds, an electrostatic interaction involving the N terminus of the peptide, and several hydrophobic interactions. This structural information, along with the binding data from BIR3 and Smac peptide mutants reported here, should aid in the design of small molecules that may be used for the treatment of cancers that overexpress IAPs.     

Structure of the 30S ribosomal subunit

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.     

Molecular structure of r(GCG)d(TATACGC): a DNA--RNA hybrid helix joined to double helical DNA

The molecule r(GCG)d(TATACGC) is self-complementary and forms two DNA--RNA hybrid segments surrounding a central region of double helical DNA; its molecular structure has been solved by X-ray analysis. All three parts of the molecule adopt a conformation which is close to that seen in the 11-fold RNA double helix. The conformation of the ribonucleotides is partly determined by water molecules bridging between the ribose O2' hydroxyl group and cytosine O2. The hybrid-DNA duplex junction contains no structural discontinuities. However, the central DNA TATA sequence has some structural irregularities.     

Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain

Short RNAs mediate gene silencing, a process associated with virus resistance, developmental control and heterochromatin formation in eukaryotes. RNA silencing is initiated through Dicer-mediated processing of double-stranded RNA into small interfering RNA (siRNA). The siRNA guide strand associates with the Argonaute protein in silencing effector complexes, recognizes complementary sequences and targets them for silencing. The PAZ domain is an RNA-binding module found in Argonaute and some Dicer proteins and its structure has been determined in the free state. Here, we report the 2.6 A crystal structure of the PAZ domain from human Argonaute eIF2c1 bound to both ends of a 9-mer siRNA-like duplex. In a sequence-independent manner, PAZ anchors the 2-nucleotide 3' overhang of the siRNA-like duplex within a highly conserved binding pocket, and secures the duplex by binding the 7-nucleotide phosphodiester backbone of the overhang-containing strand and capping the 5'-terminal residue of the complementary strand. On the basis of the structure and on binding assays, we propose that PAZ might serve as an siRNA-end-binding module for siRNA transfer in the RNA silencing pathway, and as an anchoring site for the 3' end of guide RNA within silencing effector complexes.     

Functional contacts of a transfer RNA synthetase with 2'-hydroxyl groups in the RNA minor groove.

The functional analysis of determinants on RNA has been largely limited to molecules that contain naturally occurring ribonucleotides, so little is known about the role of 2'-hydroxyl groups in protein-RNA recognition. A single base pair (G3.U70) in the acceptor stem of tRNA(Ala) is the principal element for specific recognition by Escherichia coli alanine-tRNA synthetase. This tRNA synthetase aminoacylates small RNA helices that contain the G3.U70 base pair. Furthermore, removal of the G3 exocyclic 2-amino group that projects into the minor groove eliminates aminoacylation. This 2-amino group is flanked on either side by ribose 2'-hydroxyl groups that line the minor groove. Here we use chemical synthesis to construct 32 helices that make deoxy and O-methyl substitutions of individual and multiple 2'-hydroxyl groups near and beyond the G3.U70 base pair and find that functional 2'-hydroxyl contacts are clustered within a few ?ngstroms of the critical 2-amino group. These contacts are highly specific and make a thermodynamically significant contribution to RNA recognition.     

Structural basis for transcription elongation by bacterial RNA polymerase

The RNA polymerase elongation complex (EC) is both highly stable and processive, rapidly extending RNA chains for thousands of nucleotides. Understanding the mechanisms of elongation and its regulation requires detailed information about the structural organization of the EC. Here we report the 2.5-A resolution structure of the Thermus thermophilus EC; the structure reveals the post-translocated intermediate with the DNA template in the active site available for pairing with the substrate. DNA strand separation occurs one position downstream of the active site, implying that only one substrate at a time can specifically bind to the EC. The upstream edge of the RNA/DNA hybrid stacks on the beta'-subunit 'lid' loop, whereas the first displaced RNA base is trapped within a protein pocket, suggesting a mechanism for RNA displacement. The RNA is threaded through the RNA exit channel, where it adopts a conformation mimicking that of a single strand within a double helix, providing insight into a mechanism for hairpin-dependent pausing and termination.     

Placement of protein and RNA structures into a 5 A-resolution map of the 50S ribosomal subunit.

We have calculated at 5.0 A resolution an electron-density map of the large 50S ribosomal subunit from the bacterium Haloarcula marismortui by using phases derived from four heavy-atom derivatives, intercrystal density averaging and density-modification procedures. More than 300 base pairs of A-form RNA duplex have been fitted into this map, as have regions of non-A-form duplex, single-stranded segments and tetraloops. The long rods of RNA crisscrossing the subunit arise from the stacking of short, separate double helices, not all of which are A-form, and in many places proteins crosslink two or more of these rods. The polypeptide exit channel was marked by tungsten cluster compounds bound in one heavy-atom-derivatized crystal. We have determined the structure of the translation-factor-binding centre by fitting the crystal structures of the ribosomal proteins L6, L11 and L14, the sarcin-ricin loop RNA, and the RNA sequence that binds L11 into the electron density. We can position either elongation factor G or elongation factor Tu complexed with an aminoacylated transfer RNA and GTP onto the factor-binding centre in a manner that is consistent with results from biochemical and electron microscopy studies.     

X-ray crystallography of the binding of the bacterial cell wall trisaccharide NAM-NAG-NAM to lysozyme

Hen egg white lysozyme was the first enzyme whose structure was determined by X-ray crystallography. The proposed mechanism based on this structure involves the distortion of the saccharide residue (2-acetamido-2-deoxy-D-muramic acid, NAM) in the natural substrate (an alternating beta (1 leads to 4) linked oligomer of 2-acetamido-2-deoxy-D-glucose (NAG) and NAM residues) bound to site D in the binding cleft. The importance of substrate distortion has prompted numerous enzymatic, chemical, theoretical, and physical studies, but there is little direct crystallographic evidence on the conformation of a NAM residue bound at site D. We now present the X-ray structure of the non-hydrolysed trisaccharide NAM-NAG-NAM bound in subsites B, C, D. Our interpretation of the 2.5-A resolution difference map does not involve distortion of this residue in site D. Comparison with the structure of the delta-lactone derived from tetra N-acetylchitotetraose (NAG)3NAL) bound to lysozyme suggests we may be looking at a Michaelis complex.     

Structure of the SRP19 RNA complex and implications for signal recognition particle assembly

The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein. It associates with ribosomes to mediate co-translational targeting of membrane and secretory proteins to biological membranes. In mammalian cells, the SRP consists of a 7S RNA and six protein components. The S domain of SRP comprises the 7S.S part of RNA bound to SRP19, SRP54 and the SRP68/72 heterodimer; SRP54 has the main role in recognizing signal sequences of nascent polypeptide chains and docking SRP to its receptor. During assembly of the SRP, binding of SRP19 precedes and promotes the association of SRP54 (refs 4, 5). Here we report the crystal structure at 2.3 A resolution of the complex formed between 7S.S RNA and SRP19 in the archaeon Methanococcus jannaschii. SRP19 bridges the tips of helices 6 and 8 of 7S.S RNA by forming an extensive network of direct protein RNA interactions. Helices 6 and 8 pack side by side; tertiary RNA interactions, which also involve the strictly conserved tetraloop bases, stabilize helix 8 in a conformation competent for SRP54 binding. The structure explains the role of SRP19 and provides a molecular framework for SRP54 binding and SRP assembly in Eukarya and Archaea.     

DNA cleavage catalysed by the ribozyme from Tetrahymena.

An RNA enzyme derived from the self-splicing intervening sequence of Tetrahymena thermophila catalyses sequence-specific cleavage of an oligodeoxyribonucleotide substrate. Compared with RNA, the DNA substrate is bound very weakly and is cleaved very slowly, revealing the importance of the RNA 2'-hydroxyl group in both the binding and chemical steps. The finding that catalysis by RNA can extend to DNA substrates indicates new possibilities for the transposition of intervening sequences and for the design of DNA cleavage agents with novel sequence specificities.     

Formation of a stable triplex from a single DNA strand

Homopurine.homopyrimidine DNA sequences have been shown to form triple-stranded structures readily under appropriate conditions. Interest in DNA triplexes arises from potential applications of intermolecular triplexes as antisense inhibitors of gene expression and from the possibility that intramolecular triplexes may have a role in gene expression and recombination. We recently presented NMR evidence for triplex formation from the DNA oligonucleotides d(GA)4 and d(TC)4, which showed unambiguously that the second pyrimidine strand is Hoogsteen base paired and the cytosines are protonated at N3 as required. To obtain a more well defined triplex, and to provide a model for in vivo triplex structures, we have designed and synthesized a 28-base DNA oligomer with a sequence that could potentially fold to form a triplex containing both T.A.T and C+.G.C triplets. Our NMR results indicate that the conformation at pH 5.5 is an intramolecular triplex and that a significant amount of triplex remains even at pH 8.0.     

X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation

Pentameric ligand-gated ion channels from the Cys-loop family mediate fast chemo-electrical transduction, but the mechanisms of ion permeation and gating of these membrane proteins remain elusive. Here we present the X-ray structure at 2.9 A resolution of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC) at pH 4.6 in an apparently open conformation. This cationic channel is known to be permanently activated by protons. The structure is arranged as a funnel-shaped transmembrane pore widely open on the outer side and lined by hydrophobic residues. On the inner side, a 5 A constriction matches with rings of hydrophilic residues that are likely to contribute to the ionic selectivity. Structural comparison with ELIC, a bacterial homologue from Erwinia chrysanthemi solved in a presumed closed conformation, shows a wider pore where the narrow hydrophobic constriction found in ELIC is removed. Comparative analysis of GLIC and ELIC reveals, in concert, a rotation of each extracellular beta-sandwich domain as a rigid body, interface rearrangements, and a reorganization of the transmembrane domain, involving a tilt of the M2 and M3 alpha-helices away from the pore axis. These data are consistent with a model of pore opening based on both quaternary twist and tertiary deformation.