Unusual metal specificity and structure of the group I ribozyme from Chlamydomonas reinhardtii 23S rRNA

Group I intron ribozymes require cations for folding and catalysis, and the current literature indicates that a number of cations can promote folding, but only Mg2+ and Mn2+ support both processes. However, some group I introns are active only with Mg2+, e.g. three of the five group I introns in Chlamydomonas reinhardtii. We have investigated one of these ribozymes, an intron from the 23S LSU rRNA gene of Chlamydomonas reinhardtii (Cr.LSU), by determining if the inhibition by Mn2+ involves catalysis, folding, or both. Kinetic analysis of guanosine-dependent cleavage by a Cr.LSU ribozyme, 23S.5 Delta Gb, that lacks the 3' exon and intron-terminal G shows that Mn2+ does not affect guanosine binding or catalysis, but instead promotes misfolding of the ribozyme. Surprisingly, ribozyme misfolding induced by Mn2+ is highly cooperative, with a Hill coefficient larger than that of native folding induced by Mg2+. At lower Mn2+ concentrations, metal inhibition is largely alleviated by the guanosine cosubstrate (GMP). The concentration dependence of guanosine cosubstrate-induced folding suggests that it functions by interacting with the G binding site, perhaps by displacing an inhibitory Mn2+. Because of these and other properties of Cr.LSU, the tertiary structure of the intron from 23S.5 Delta Gb was examined using Fe2+-EDTA cleavage. The ground-state structure shows evidence of an unusually open ribozyme core: the catalytic P3-P7 domain and the nucleotides that connect it to the P4-P5-P6 domain are exposed to solvent. The implications of this structure for the in vitro and in vivo properties of this intron ribozyme are discussed.     

RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis

The group I intron has served as a model for RNA catalysis since its discovery 25 years ago. Four recently determined high-resolution crystal structures complement extensive biochemical studies on this system. Structures of the Azoarcus, Tetrahymena and bacteriophage Twort group I introns mimic different states of the splicing or ribozyme reaction pathway and provide information on splice site selection and metal ion catalysis. The 5'-splice site is selected by formation of a conserved G.U wobble pair between the 5'-exon terminus and the intron. The 3'-splice site is identified through stacking of three base triples, in which the middle triple contains the conserved terminal nucleotide of the intron, OmegaG. The structures support a two-metal-ion mechanism for group I intron splicing that might have corollaries to group II intron and pre-mRNA splicing by the spliceosome.     

The 2'-hydroxyl group of the guanosine nucleophile donates a functionally important hydrogen bond in the tetrahymena ribozyme reaction

In the first step of self-splicing, group I introns utilize an exogenous guanosine nucleophile to attack the 5'-splice site. Removal of the 2'-hydroxyl of this guanosine results in a 10 (6)-fold loss in activity, indicating that this functional group plays a critical role in catalysis. Biochemical and structural data have shown that this hydroxyl group provides a ligand for one of the catalytic metal ions at the active site. However, whether this hydroxyl group also engages in hydrogen-bonding interactions remains unclear, as attempts to elaborate its function further usually disrupt the interactions with the catalytic metal ion. To address the possibility that this 2'-hydroxyl contributes to catalysis by donating a hydrogen bond, we have used an atomic mutation cycle to probe the functional importance of the guanosine 2'-hydroxyl hydrogen atom. This analysis indicates that, beyond its role as a ligand for a catalytic metal ion, the guanosine 2'-hydroxyl group donates a hydrogen bond in both the ground state and the transition state, thereby contributing to cofactor recognition and catalysis by the intron. Our findings continue an emerging theme in group I intron catalysis: the oxygen atoms at the reaction center form multidentate interactions that function as a cooperative network. The ability to delineate such networks represents a key step in dissecting the complex relationship between RNA structure and catalysis.     

Structure of the Tetrahymena ribozyme: base triple sandwich and metal ion at the active site

The Tetrahymena intron is an RNA catalyst, or ribozyme. As part of its self-splicing reaction, this ribozyme catalyzes phosphoryl transfer between guanosine and a substrate RNA strand. Here we report the refined crystal structure of an active Tetrahymena ribozyme in the absence of its RNA substrate at 3.8 A resolution. The 3'-terminal guanosine (omegaG), which serves as the attacking group for RNA cleavage, forms a coplanar base triple with the G264-C311 base pair, and this base triple is sandwiched by three other base triples. In addition, a metal ion is present in the active site, contacting or positioned close to the ribose of the omegaG and five phosphates. All of these phosphates have been shown to be important for catalysis. Therefore, we provide a picture of how the ribozyme active site positions both a catalytic metal ion and the nucleophilic guanosine for catalysis prior to binding its RNA substrate.     

Distinct sites of phosphorothioate substitution interfere with folding and splicing of the Anabaena group I intron

Although the active site of group I introns is phylogenetically conserved, subclasses of introns have evolved different mechanisms of stabilizing the catalytic core. Large introns contain weakly conserved 'peripheral' domains that buttress the core through predicted interhelical contacts, while smaller introns use loop-helix interactions for stability. In all cases, specific and non-specific magnesium ion binding accompanies folding into the active structure. Whether similar RNA-RNA and RNA-magnesium ion contacts play related functional roles in different introns is not clear, particularly since it can be difficult to distinguish interactions directly involved in catalysis from those important for RNA folding. Using phosphorothioate interference with RNA activity and structure in the small (249 nt) group I intron from Anabaena, we used two independent assays to detect backbone phosphates important for catalysis and those involved in intron folding. Comparison of the interference sites identified in each assay shows that positions affecting catalysis cluster primarily in the conserved core of the intron, consistent with conservation of functionally important phosphates, many of which are magnesium ion binding sites, in diverse group I introns, including those from Azoarcus and Tetrahymena. However, unique sites of folding interference located outside the catalytic core imply that different group I introns, even within the same subclass, use distinct sets of tertiary interactions to stabilize the structure of the catalytic core.     

Analysis of the P7 region within the catalytic core of the Tetrahymena ribozyme by employing in vitro selection.

The highly conserved P7 region is generally believed to act as a major portion of the catalytic site in the Group I intron ribozyme. However, its functions have not been elucidated except for the fact that it specifically binds a cofactor guanosine required for self-splicing reaction. We attempted an in vitro selection experiment to determine the sequence requirements of this region in the mechanism of catalysis by using the Tetrahymena ribozyme. We found that the selected active clones have the secondary structure similar to that of the wild type with few exceptions. However, their primary sequences were not conserved except G264 and C311 that are the major elements of the binding site for the guanosine. Our results suggest that the unique secondary structure of the P7 region is a primary requisite for the catalytic function of this class of ribozymes.     

Streptomycin inhibits splicing of group I introns by competition with the guanosine substrate.

Streptomycin is an aminocyclitol glycoside antibiotic, which interferes with prokaryotic protein synthesis by interacting with the ribosomal RNA. We report here that streptomycin is also able to inhibit self splicing of the group I intron of the thymidylate synthase gene of phage T4. The inhibition is kinetically competitive with the substrate guanosine. Streptomycin and guanosine have in common a guanidino group, which has been shown to undergo hydrogen bonds with the ribozyme (Bass & Cech, Biochemistry, 25, 1986, 4473). The inhibitory effect of streptomycin extends to other group I introns, but does not affect group II introns. Mutating the bulged nucleotide in the conserved P7 secondary structure element of the td intron alters the affinity of the ribozyme for both guanosine and streptomycin. Myomycin, an antibiotic with similar effects on protein synthesis as streptomycin, is also able to inhibit splicing. In contrast, bluensomycin, which is structurally related to streptomycin, but contains only one guanidino group does not inhibit splicing. We discuss these findings in support of an evolutionary model that stresses the antiquity of antibiotics (J. Davies, Molecular Microbiology 4, 1990, 1227).     

Inhibition of self-splicing group I intron RNA: high-throughput screening assays.

High-throughput screening assays have been developed to rapidly identify small molecule inhibitors targeting catalytic group I introns. Biochemical reactions catalyzed by a self-splicing group I intron derived from Pneumocystis carinii or from bacteriophage T4 have been investigated. In vitro biochemical assays amenable to high-throughput screening have been established. Small molecules that inhibit the functions of group I introns have been identified. These inhibitors should be useful in better understanding ribozyme catalysis or in therapeutic intervention of group I intron-containing microorganisms.     

The P5abc peripheral element facilitates preorganization of the tetrahymena group I ribozyme for catalysis

Phylogenetic comparisons and site-directed mutagenesis indicate that group I introns are composed of a catalytic core that is universally conserved and peripheral elements that are conserved only within intron subclasses. Despite this low overall conservation, peripheral elements are essential for efficient splicing of their parent introns. We have undertaken an in-depth structure-function analysis to investigate the role of one of these elements, P5abc, using the well-characterized ribozyme derived from the Tetrahymena group I intron. Structural comparisons using solution-based free radical cleavage revealed that a ribozyme lacking P5abc (E(DeltaP5abc)) and E(DeltaP5abc) with P5abc added in trans (E(DeltaP5abc).P5abc) adopt a similar global tertiary structure at Mg(2+) concentrations greater than 20 mM [Doherty, E. A., et al. (1999) Biochemistry 38, 2982-90]. However, free E(DeltaP5abc) is greatly compromised in overall oligonucleotide cleavage activity, even at Mg(2+) concentrations as high as 100 mM. Further characterization of E(DeltaP5abc) via DMS modification revealed local structural differences at several positions in the conserved core that cluster around the substrate binding sites. Kinetic and thermodynamic dissection of individual reaction steps identified defects in binding of both substrates to E(DeltaP5abc), with > or =25-fold weaker binding of a guanosine nucleophile and > or =350-fold weaker docking of the oligonucleotide substrate into its tertiary interactions with the ribozyme core. These defects in binding of the substrates account for essentially all of the 10(4)-fold decrease in overall activity of the deletion mutant. Together, the structural and functional observations suggest that the P5abc peripheral element not only provides stability but also positions active site residues through indirect interactions, thereby preferentially stabilizing the active ribozyme structure relative to alternative less active states. This is consistent with the view that peripheral elements engage in a network of mutually reinforcing interactions that together ensure cooperative folding of the ribozyme to its active structure.     

Leaving group stabilization by metal ion coordination and hydrogen bond donation is an evolutionarily conserved feature of group I introns.

To understand the behavior of group I introns on a biologically fundamental level, we must distinguish those traits that arise as the products of natural selection (selected traits) from those that arise as the products of neutral drift (non-selected traits). In practice, this distinction relies on comparing the similarities and differences among widely divergent introns to identify conserved traits. Here we address whether the strategies used by the eukaryotic group I intron from the Tetrahymena ciliate to stabilize the leaving group during splicing are maintained in the group I intron from the widely divergent Azoarcus bacterium. A substrate analogue containing a 3'-phosphorothiolate linkage, in which a sulfur atom replaces the bridging 3'-oxygen atom of the scissile phosphate, reacts 20-fold slower in the Azoarcus reaction than the corresponding unmodified substrate in the presence of Mg(II) as the only divalent cation. However, Mn(II) relieves this negative effect such that the 3'-S-P bond cleaves 21-fold faster than does the 3'O-P bond. Other thiophilic divalent metal ions such as Co(II), Cd(II), and Zn(II) similarly support cleavage of the S-P bond. These results indicate that a metal ion directly coordinates to the leaving group in the transition state of the Azoarcus ribozyme reaction. Additionally, the 3'-sulfur substitution eliminates the approximately 10(3)-fold contribution of the adjacent 2'-OH to transition state stabilization. Considering that sulfur accepts hydrogen bonds weakly compared to oxygen, this result suggests that the 2'-OH contributes to catalysis by donating a hydrogen bond to the 3'-oxygen leaving group in the transition state, presumably acting in conjunction with the metal ion to stabilize the developing negative charge. These same catalytic strategies of metal ion coordination and hydrogen bond donation operate in the Tetrahymena ribozyme reaction, suggesting that these features of catalysis have been conserved during evolution and thus extend to all group I introns. The two ribozymes also exhibit quantitative differences in their response to 3'-sulfur substitution. The Azoarcus ribozyme binds and cleaves the phosphorothiolate substrate more efficiently relative to the natural substrate than the Tetrahymena ribozyme under the same conditions, suggesting that the Azoarcus ribozyme better accommodates the phosphorothiolate at the active site both in the ground state and in the transition state. These differences may reflect either a less tightly knit Azoarcus structure and/or spatial deviations between backbone atoms in the two ribozymes that arise during divergent evolution, analogous to the well-documented relationship between protein sequence and structure.     

Domains 2 and 3 interact to form critical elements of the group II intron active site

Group II introns are self-splicing RNA molecules that also behave as mobile genetic elements. The secondary structure of group II intron RNAs is typically described as a series of six domains that project from a central wheel. Most structural and mechanistic analyses of the intron have focused on domains 1 and 5, which contain the residues essential for catalysis, and on domain 6, which contains the branch-point adenosine. Domains 2 and 3 (D2, D3) have been shown to make important contributions to intronic activity; however, information about their function is quite limited. To elucidate the role of D2 and D3 in group II ribozyme catalysis, we built a series of multi-piece ribozyme constructs based on the ai5gamma group II intron. These constructs are designed to shed light on the roles of D2 and D3 in some of the major reactions catalyzed by the intron: 5'-exon cleavage, branching, and substrate hydrolysis. Reactions with these constructs demonstrate that D3 stimulates the chemical rate constant of group II intron reactions, and that it behaves as a form of catalytic effector. However, D3 is unable to associate independently with the ribozyme core. Docking of D3 is mediated by a short duplex that is found at the base of D2. In addition to recruiting D3 into the core, the D2 stem directs the folding of the adjacent j(2/3) linker, which is among the most conserved elements in the group II intron active site. In turn, the D2 stem contributes to 5'-splice site docking and ribozyme conformational change. Nucleotide analog interference mapping suggests an interaction between the D2 stem and D3 that builds on the known theta-theta' interaction and extends it into D3. These results establish that D3 and the base of D2 are key elements of the group II intron core and they suggest a hierarchy for active-site assembly.     

Metal ion binding sites in a group II intron core

Group II introns are catalytic RNA molecules that require divalent metal ions for folding, substrate binding, and chemical catalysis. Metal ion binding sites in the group II core have now been elucidated by monitoring the site-specific RNA hydrolysis patterns of bound ions such as Tb(3+) and Mg(2+). Major sites are localized near active site elements such as domain 5 and its surrounding tertiary interaction partners. Numerous sites are also observed at intron substructures that are involved in binding and potentially activating the splice sites. These results highlight the locations of specific metal ions that are likely to play a role in ribozyme catalysis.     

The pseudodisaccharides: a novel class of group I intron splicing inhibitors.

Lysinomicin, a naturally-occurring pseudodisaccharide, inhibits translation in prokaryotes. We report that lysinomicin (and three related compounds) are able to inhibit the self-splicing of group I introns, thus identifying pseudodisaccharides as a novel class of group I intron splicing inhibitors. Lysinomicin inhibited the self-splicing of the sunY intron of phage T4 with a Ki of 8.5 microM (+/- 5 microM) and was active against other group I introns. Inhibition was found to be competitive with the substrate guanosine, unlike aminoglycoside antibiotics, which act non-competitively to inhibit the splicing of group I introns. Competitive inhibitors of group I intron splicing known to date all contain a guanidino group that was thought to be required for inhibition; lysinomicin lacks a guanidino group.     

Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing.

We have characterized the structural organization and catalytic properties of the large nucleolar group I introns (NaSSU1) of the different Naegleria species N. jamiesoni, N. andersoni, N. italica, and N. gruberi. NaSSU1 consists of three distinct RNA domains: an open reading frame encoding a homing-type endonuclease, and a small group I ribozyme (NaGIR1) inserted into the P6 loop of a second group I ribozyme (NaGIR2). The two ribozymes have different functions in RNA splicing and processing. NaGIR1 is an unusual self-cleaving group I ribozyme responsible for intron processing at two internal sites (IPS1 and IPS2), both close to the 5' end of the open reading frame. This processing is hypothesized to lead to formation of a messenger RNA for the endonuclease. Structurally, NaGIR2 is a typical group IC1 ribozyme, catalyzing intron excision and exon ligation reactions. NaGIR2 is responsible for circularization of the excised intron, a reaction that generates full-length RNA circles of wild-type intron. Although it is only distantly related in primary sequence, NaSSU1 RNA has a predicted organization and function very similar to that of the mobile group I intron DiSSU1 of Didymium, the only other group I intron known to encode two ribozymes. We propose that these twin-ribozyme introns define a distinct category of group I introns with a conserved structural organization and function.     

Productive folding to the native state by a group II intron ribozyme.

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5gamma. The isotherms span a similar range of Mg(2+) concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5gamma intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5gamma occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.     

A chemical phylogeny of group I introns based upon interference mapping of a bacterial ribozyme

Despite its small size, the 205 nt group I intron from Azoarcus tRNA(Ile) is an exceptionally stable self-splicing RNA. This IC3 class intron retains the conserved secondary structural elements common to group I ribozymes, but lacks several peripheral helices. These features make it an ideal system to establish the conserved chemical basis of group I intron activity. We collected nucleotide analog interference mapping (NAIM) data of the Azoarcus intron using 14 analogs that modified the phosphate backbone, the ribose sugar, or the purine base functional groups. In conjunction with a complete interference set collected on the Tetrahymena group I intron (IC1 class), these data define a "chemical phylogeny" of functional groups that are important for the activity of both introns and that may be common chemical features of group I intron catalysts. The data identify the functional moieties most likely to play a conserved role as ligands for catalytic metal ions, the substrate helix, and the guanosine cofactor. These include backbone functional groups whose nucleotide identity is not conserved, and hence are difficult to identify by standard phylogenetic sequence comparisons. The data suggest that both introns utilize an equivalent set of long range tertiary interactions for 5'-splice site selection between the P1 substrate helix and its receptor in the J4/5 asymmetric bulge, as well as an equivalent set of 2'-OH groups for P1 helix docking into most of the single stranded segment J8/7. However, the Azoarcus intron appears to make an alternative set of interactions at the base of the P1 helix and at the 5'-end of the J8/7. Extensive differences were observed within the intron peripheral domains, particularly in P2 and P8 where the Azoarcus data strongly support the proposed formation of a tetraloop-tetraloop receptor interaction. This chemical phylogeny for group I intron catalysis helps to refine structural models of the RNA active site and identifies functional groups that should be carefully investigated for their role in transition state stabilization.     

Mapping divalent metal ion binding sites in a group II intron by Mn(2+)- and Zn(2+)-induced site-specific RNA cleavage.

The function of group II introns depends on positively charged divalent metal ions that stabilize the ribozyme structure and may be directly involved in catalysis. We investigated Mn2+- and Zn2+-induced site-specific RNA cleavage to identify metal ions that fit into binding pockets within the structurally conserved bI1 group II intron domains (DI-DVI), which might fulfill essential roles in intron function. Ten cleavage sites were identified in DI, two sites in DIII and two in DVI. All cleavage sites are located in the center or close to single-stranded and flexible RNA structures. Strand scissions mediated by Mn2+/Zn2+ are competed for by Mg2+, indicating the existence of Mg2+ binding pockets in physical proximity to the observed Mn2+-/Zn2+-induced cleavage positions. To distinguish between metal ions with a role in structure stabilization and those that play a more specific and critical role in the catalytic process of intron splicing, we combined structural and functional assays, comparing wild-type precursor and multiple splicing-deficient mutants. We identified six regions with binding pockets for Mg2+ ions presumably playing an important role in bI1 structure stabilization. Remarkably, assays with DI deletions and branch point mutants revealed the existence of one Mg2+ binding pocket near the branching A, which is involved in first-step catalysis. This pocket formation depends on precise interaction between the branching nucleotide and the 5' splice site, but does not require exon-binding site 1/intron binding site 1 interaction. This Mg2+ ion might support the correct placing of the branching A into the 'first-step active site'.     

A glimpse into the active site of a group II intron and maybe the spliceosome, too

The X-ray crystal structure of an excised group II self-splicing intron was recently solved by the Pyle group. Here we review some of the notable features of this structure and what they may tell us about the catalytic active site of the group II ribozyme and potentially the spliceosome. The new structure validates the central role of domain V in both the structure and catalytic function of the ribozyme and resolves several outstanding puzzles raised by previous biochemical, genetic and structural studies. While lacking both exons as well as the cleavage sites and nucleophiles, the structure reveals how a network of tertiary interactions can position two divalent metal ions in a configuration that is ideal for catalysis.