Stereochemical course of catalysis by the Tetrahymena ribozyme.

The group I intron from Tetrahymena catalyzes phosphodiester transfer reactions on various RNA substrates. A modified RNA substrate with a phosphorothioate group in one stereoisomeric form at the site of reaction was synthesized in order to determine the stereochemical course of an RNA-catalyzed reaction. The reaction product was digested with a stereospecific nuclease to determine the configuration of the product phosphorothioate. The reaction occurs with inversion of configuration at phosphorus, implying an in-line pathway for the reaction.

A multisubunit ribozyme that is a catalyst of and template for complementary strand RNA synthesis.

Derivatives of the sunY self-splicing intron efficiently catalyzed the synthesis of complementary strand RNA by template-directed assembly of oligonucleotides. These ribozymes were separated into three short RNA fragments that formed active catalytic complexes. One of the multisubunit sunY derivatives catalyzed the synthesis of a strand of RNA complementary to one of its own subunits. These results suggest that prebiotically synthesized oligonucleotides might have been able to assemble into a complex capable of self-replication.

Crystal structure of the ribonucleoprotein core of the signal recognition particle.

The signal recognition particle (SRP), a protein-RNA complex conserved in all three kingdoms of life, recognizes and transports specific proteins to cellular membranes for insertion or secretion. We describe here the 1.8 angstrom crystal structure of the universal core of the SRP, revealing protein recognition of a distorted RNA minor groove. Nucleotide analog interference mapping demonstrates the biological importance of observed interactions, and genetic results show that this core is functional in vivo. The structure explains why the conserved residues in the protein and RNA are required for SRP assembly and defines a signal sequence recognition surface composed of both protein and RNA.

Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit.

Initiation of protein synthesis in eukaryotes requires recruitment of the 40S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5' end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5' untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40S ribosomal subunit at about 20 A resolution. IRES binding induces a pronounced conformational change in the 40S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.

Crystal structure of a group I ribozyme domain: principles of RNA packing.

Group I self-splicing introns catalyze their own excision from precursor RNAs by way of a two-step transesterification reaction. The catalytic core of these ribozymes is formed by two structural domains. The 2.8-angstrom crystal structure of one of these, the P4-P6 domain of the Tetrahymena thermophila intron, is described. In the 160-nucleotide domain, a sharp bend allows stacked helices of the conserved core to pack alongside helices of an adjacent region. Two specific long-range interactions clamp the two halves of the domain together: a two-Mg2+-coordinated adenosine-rich corkscrew plugs into the minor groove of a helix, and a GAAA hairpin loop binds to a conserved 11-nucleotide internal loop. Metal- and ribose-mediated backbone contacts further stabilize the close side-by-side helical packing. The structure indicates the extent of RNA packing required for the function of large ribozymes, the spliceosome, and the ribosome.

RNA tertiary structure mediation by adenosine platforms.

The crystal structure of a group I intron domain reveals an unexpected motif that mediates both intra- and intermolecular interactions. At three separate locations in the 160-nucleotide domain, adjacent adenosines in the sequence lie side-by-side and form a pseudo-base pair within a helix. This adenosine platform opens the minor groove for base stacking or base pairing with nucleotides from a noncontiguous RNA strand. The platform motif has a distinctive chemical modification signature that may enable its detection in other structured RNAs. The ability of this motif to facilitate higher order folding provides one explanation for the abundance of adenosine residues in internal loops of many RNAs.

RNA seeing double: close-packing of helices in RNA tertiary structure.

Structured RNA molecules play essential roles in RNA processing, chromosome maintenance and protein biosynthesis. RNA necessarily uses different strategies than proteins for folding and assembly of complex architectures. The RNA-folding problem is largely an issue of helical packing: how does RNA organize and pack short, double-helical segments to produce active sites and recognition motifs for proteins? Noncanonical base pairs, metal ions and 2'-hydroxyl groups are key elements in RNA higher-order structure formation.

Ribozymes: the hammerhead swings into action.

A new crystal structure of a modified hammerhead ribozyme reveals an intermediate conformation that may explain discrepancies between previous structures and the required orientation of the labile bond in the ribozyme's active site.

Cashing in on crystals.

Structures of the ribosome and its two subunits have been determined by X-ray crystallography at resolutions sufficient to reveal interactions between RNA and protein in the subunits and orientations of substrates at the subunit interface in the intact ribosome.

Miniribozymes, small derivatives of the sunY intron, are catalytically active.

The self-splicing sunY intron from bacteriophage T4 has the smallest conserved core secondary structure of any of the active group I introns. Here we show that several nonconserved regions can be deleted from this intron without complete loss of catalytic activity. The 3' stems P9, P9.1, and P9.2 can be deleted while retaining 5' cleaving activity. Two base-paired stems (P7.1 and P7.2) that are peculiar to the group IA introns can also be deleted; however, the activities of the resulting derivatives depend greatly on the choice of replacement sequences and their lengths. The smallest active derivative is less than 180 nucleotides long. These experiments help to define the minimum structural requirements for catalysis.

Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

The Tetrahymena ribozyme has been shown to catalyze an RNA polymerase-like reaction in which an RNA primer is extended by the sequential addition of pN nucleotides derived from GpN dinucleotides, where N = A, C, or U. Here, we show that this reaction is influenced by the presence of a template; bases that can form Watson-Crick base pairs with a template add as much as 25-fold more efficiently than mismatched bases. A mutant enzyme with an altered guanosine binding site can catalyze template-directed primer extension with all four bases when supplied with dinucleotides of the form 2-aminopurine-pN.

RNA structure: crystal clear?

Structured RNAs play an essential role in chromosome maintenance, RNA processing, protein biosynthesis, and protein transport. To understand RNA function in these diverse biological systems, the rules for RNA folding and recognition must be learned. Recent crystal structures of hammerhead ribozymes, a group I intron domain, and RNA duplexes provide new insights into the principles of RNA folding and function.

Hammerhead ribozyme structure: U-turn for RNA structural biology.

Two crystal structures of the hammerhead ribozyme provide the first atomic-resolution views of an RNA active site, and suggest that the catalytic center may reside in a U-turn motif which was first seen in tRNA(Phe).

Metal-binding sites in the major groove of a large ribozyme domain.

BACKGROUND: Group I self-splicing introns catalyze sequential transesterification reactions within an RNA transcript to produce the correctly spliced product. Often several hundred nucleotides in size, these ribozymes fold into specific three-dimensional structures that confer activity. The 2.8 A crystal structure of a central component of the Tetrahymena thermophila group I intron, the 160-nucleotide P4-P6 domain, provides the first detailed view of metal binding in an RNA large enough to exhibit side-by-side helical packing. The long-range contacts and bound ligands that stabilize this fold can now be examined in detail. RESULTS: Heavy-atom derivatives used for the structure determination reveal characteristics of some of the metal-binding sites in the P4-P6 domain. Although long-range RNA-RNA contacts within the molecule primarily involve the minor groove, osmium hexammine binds at three locations in the major groove. All three sites involve G and U nucleotides exclusively; two are formed by G.U wobble base pairs. In the native RNA, two of the sites are occupied by fully-hydrated magnesium ions. Samarium binds specifically to the RNA by displacing a magnesium ion in a region critical to the folding of the entire domain. CONCLUSIONS: Bound at specific sites in the P4-P6 domain RNA, osmium (III) hexammine produced the high-quality heavy-atom derivative used for structure determination. These sites can be engineered into other RNAs, providing a rational means of obtaining heavy-atom derivatives with hexammine compounds. The features of the observed metal-binding sites expand the known repertoire of ligand-binding motifs in RNA, and suggest that some of the conserved tandem G.U base pairs in ribosomal RNAs are magnesium-binding sites.

Metal ions in ribozyme folding and catalysis.

Current research is reshaping basic theories regarding the roles of metal ions in ribozyme function. No longer viewed as strict metalloenzymes, some ribozymes can access alternative catalytic mechanisms depending on the identity and availability of metal ions. Similarly, reaction conditions can allow different folding pathways to predominate, with divalent cations sometimes playing opposing roles.

Crystallization and structure determination of a hepatitis delta virus ribozyme: use of the RNA-binding protein U1A as a crystallization module.

Well-ordered crystals of a genomic hepatitis delta virus (HDV) ribozyme, a large, globular RNA, were obtained employing a new crystallization method. A high-affinity binding site for the spliceosomal protein U1A was engineered into a segment of the catalytic RNA that is dispensable for catalysis. Because molecular surfaces of proteins are more chemically varied than those of RNA, the presence of the protein moiety was expected to facilitate crystallization and improve crystal order. The HDV ribozyme-U1A complex crystallized readily, and its structure was solved using standard techniques for heavy-atom derivatization of protein crystals. Over 1200 A(2) of the solvent-accessible surface area of the complex are involved in crystal contacts. As protein-protein interactions comprise 85% of this buried area, these crystals appear to be held together predominantly by the protein component of the complex. Our crystallization method should be useful for the structure determination of other biochemically important RNAs for which protein partners do not exist or are experimentally intractable. The refined model of the complex (R-free=27.9% for all reflections between 20.0 and 2.3 A) reveals an RNA with a deep active site cleft. Well-ordered metal ions are not observed crystallographically in this cavity. Biochemical results of previous workers had suggested an important role in catalysis for cytosine 75. The pyrimidine base of this residue is buried at the bottom of the active site in an environment that could raise its pK(a) value. We propose that this highly conserved cytosine may be the general base that catalyzes the transesterification.

A general module for RNA crystallization.

Crystallization of RNA molecules other than simple oligonucleotide duplexes remains a challenging step in structure determination by X-ray crystallography. Subjecting biochemically, covalently and conformationally homogeneous target molecules to an exhaustive array of crystallization conditions is often insufficient to yield crystals large enough for X-ray data collection. Even when large RNA crystals are obtained, they often do not diffract X-rays to resolutions that would lead to biochemically informative structures. We reasoned that a well-folded RNA molecule would typically present a largely undifferentiated molecular surface dominated by the phosphate backbone. During crystal nucleation and growth, this might result in neighboring molecules packing subtly out of register, leading to premature crystal growth cessation and disorder. To overcome this problem, we have developed a crystallization module consisting of a normally intramolecular RNA-RNA interaction that is recruited to make an intermolecular crystal contact. The target RNA molecule is engineered to contain this module at sites that do not affect biochemical activity. The presence of the crystallization module appears to drive crystal growth, in the course of which other, non-designed contacts are made. We have employed the GAAA tetraloop/tetraloop receptor interaction successfully to crystallize numerous group II intron domain 5-domain 6, and hepatitis delta virus (HDV) ribozyme RNA constructs. The use of the module allows facile growth of large crystals, making it practical to screen a large number of crystal forms for favorable diffraction properties. The method has led to group II intron domain crystals that diffract X-radiation to 3.5 A resolution.

Structural and energetic analysis of RNA recognition by a universally conserved protein from the signal recognition particle.

The signal recognition particle (SRP) is a ribonucleoprotein complex responsible for targeting proteins to the endoplasmic reticulum in eukarya or to the inner membrane in prokarya. The crystal structure of the universally conserved RNA-protein core of the Escherichia coli SRP, refined here to 1.5 A resolution, revealed minor groove recognition of the 4.5 S RNA component by the M domain of the Ffh protein. Within the RNA, nucleotides comprising two phylogenetically conserved internal loops create a unique surface for protein recognition. To determine the energetic importance of conserved nucleotides for SRP assembly, we measured the affinity of the M domain for a series of RNA mutants. This analysis reveals how conserved nucleotides within the two internal loop motifs establish the architecture of the macromolecular interface and position essential functional groups for direct recognition by the protein.

The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold.

Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) located in the 5' untranslated region of the genomic RNA that drives cap-independent initiation of translation of the viral message. The approximate secondary structure and minimum functional length of the HCV IRES are known, and extensive mutagenesis has established that nearly all secondary structural domains are critical for activity. However, the presence of an IRES RNA tertiary fold and its functional relevance have not been established. Using chemical and enzymatic probes of the HCV IRES RNA in solution, we show that the IRES adopts a unique three-dimensional structure at physiological salt concentrations in the absence of additional cofactors or the translation apparatus. Folding of the IRES involves cooperative uptake of magnesium and is driven primarily by charge neutralization. This tertiary structure contains at least two independently folded regions which closely correspond to putative binding sites for the 40 S ribosomal subunit and initiation factor 3 (eIF3). Point mutations that inhibit IRES folding also inhibit its function, suggesting that the IRES tertiary structure is essential for translation initiation activity. Chemical and enzymatic probing data and small-angle X-ray scattering (SAXS) experiments in solution show that upon folding, the IRES forms an extended structure in which functionally important loops are exposed. These results suggest that the 40 S ribosomal subunit and eIF3 bind an HCV IRES that is prefolded to spatially organize recognition domains.

Mutational analysis of conserved nucleotides in a self-splicing group I intron.

We have constructed all single base substitutions in almost all of the highly conserved residues of the Tetrahymena self-splicing intron. Mutation of highly conserved residues almost invariably leads to loss of enzymatic activity. In many cases, activity could be regained by making additional mutations that restored predicted base-pairings; these second site suppressors in general confirm the secondary structure derived from phylogenetic data. At several positions, our suppression data can be most readily explained by assuming non-Watson-Crick base-pairings. In addition to the requirements imposed by the secondary structure, the sequence of the intron is constrained by "negative interactions", the exclusion of particular nucleotide sequences that would form undesirable secondary structures. A comparison of genetic and phylogenetic data suggests sites that may be involved in tertiary structural interactions.