Conserved base-pairings between C266-A268 and U307-G309 in the P7 of the Tetrahymena ribozyme is nonessential for the in vitro self-splicing reaction.

P7 is highly conserved in Group I self-splicing intron ribozymes. This region is known to coordinate metal ions and bind a cofactor guanosine required for the self-splicing. To further investigate the fundamental role of the corresponding region in the Tetrahymena ribozyme, we attempted to identify minimal requirements for the base-paired region excluding the guanosine binding site. We discovered that a variety of sequences are eligible and its derivatives possessing extra nucleotide(s) can still conduct the first step of splicing, indicating that no particular base-pairing is essential in this region for conducting the reaction in vitro. The results provide two hypotheses for the fundamental role of this region: (i) if the region contains element(s) that are strictly required in the catalysis, they are not necessarily tightly fixed in the ribozyme and (ii) if not, its fundamental role may simply be to coordinate neighboring regions that are directly involved in the catalysis.     

Structure-function analysis from the outside in: long-range tertiary contacts in RNA exhibit distinct catalytic roles

The conserved catalytic core of the Tetrahymena group I ribozyme is encircled by peripheral elements. We have conducted a detailed structure-function study of the five long-range tertiary contacts that fasten these distal elements together. Mutational ablation of each of the tertiary contacts destabilizes the folded ribozyme, indicating a role of the peripheral elements in overall stability. Once folded, three of the five tertiary contact mutants exhibit defects in overall catalysis that range from 20- to 100-fold. These and the subsequent results indicate that the structural ring of peripheral elements does not act as a unitary element; rather, individual connections have distinct roles as further revealed by kinetic and thermodynamic dissection of the individual reaction steps. Ablation of P14 or the metal ion core/metal ion core receptor (MC/MCR) destabilizes docking of the substrate-containing P1 helix into tertiary interactions with the ribozyme's conserved core. In contrast, ablation of the L9/P5 contact weakens binding of the guanosine nucleophile by slowing its association, without affecting P1 docking. The P13 and tetraloop/tetraloop receptor (TL/TLR) mutations had little functional effect and small, local structural changes, as revealed by hydroxyl radical footprinting, whereas the P14, MC/MCR, and L9/P5 mutants show structural changes distal from the mutation site. These changes extended into regions of the catalytic core involved in docking or guanosine binding. Thus, distinct allosteric pathways couple the long-range tertiary contacts to functional sites within the conserved core. This modular functional specialization may represent a fundamental strategy in RNA structure-function interrelationships.     

Relationship between the self-splicing activity and the solidity of the master domain of the Tetrahymena group I ribozyme

The highly conserved P3-P7 domain of the Group I intron ribozymes is known to contain essential elements, such as the binding site for the cofactor guanosine, required for conducting the splicing reaction. We investigated the domain of the Tetrahymena intron ribozyme and its variants in order to clarify the relationship between its stability and function. We found that the destabilization of the P3-P7 domain facilitates the active structure formation at high magnesium ion concentrations where the formation is retarded for the wild type. The destabilized domain also increases K(GTP)(m) although this can be compensated by increasing the concentration of Mg(2+), indicating that the stable domain is required for establishing a tight guanosine binding site. The results suggest that the stability of the domain affects the rate-limiting step in the RNA folding pathway and also regulates the efficiency of the splicing reaction.     

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.     

Crystal structure of a phage Twort group I ribozyme-product complex

Group I introns are catalytic RNAs capable of orchestrating two sequential phosphotransesterification reactions that result in self-splicing. To understand how the group I intron active site facilitates catalysis, we have solved the structure of an active ribozyme derived from the orf142-I2 intron from phage Twort bound to a four-nucleotide product RNA at a resolution of 3.6 A. In addition to the three conserved domains characteristic of all group I introns, the Twort ribozyme has peripheral insertions characteristic of phage introns. These elements form a ring that completely envelops the active site, where a snug pocket for guanosine is formed by a series of stacked base triples. The structure of the active site reveals three potential binding sites for catalytic metals, and invokes a role for the 2' hydroxyl of the guanosine substrate in organization of the active site for catalysis.     

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).     

Molecular recognition in a trans excision-splicing ribozyme: non-Watson-Crick base pairs at the 5' splice site and omegaG at the 3' splice site can play a role in determining the binding register of reaction substrates

Trans excision-splicing (TES) ribozymes, derived from a Pneumocystis carinii group I intron, can catalyze the excision of targeted sequences from within RNAs. In this report, the sequence requirements of the splice sites are analyzed. These conserved sequences include a u-G wobble pair at the 5' splice site and a guanosine in the omega position at the 3' splice site (in the substrate). We report that 7 out of 16 base pair combinations at the 5' splice site produce appreciable TES product. This promiscuity is in contrast to results reported for analogous self-splicing reactions using a Tetrahymena ribozyme. At long reaction times TES products dissociate and rebind free ribozyme, at which point product degradation occurs via the 5' cleavage reaction. Unexpectedly, only in cases where Watson-Crick base pairs form at the 5'splice site do we see degradation of TES products at cryptic sites, suggesting that non-Watson-Crick base pairs at the 5' splice site are acting in concert with other factors to precisely determine the binding register of TES reaction substrates within the ribozyme. Moreover, cryptic site degradation does not occur with the corresponding reaction substrates, which additionally contain omegaG, suggesting that omegaG can play a similar role. We report that omegaG cannot be replaced by any other base, so TES substrates require a guanosine as the last (or only) base to be excised. Additionally, we demonstrate that P9.0 and P10 are expendable for TES reactions, suggesting that omegaG is sufficient as a 3' molecular recognition element.     

Identification of phosphates involved in catalysis by the ribozyme RNase P RNA.

The RNA subunit of ribonuclease P (RNase P RNA) is a catalytic RNA that cleaves precursor tRNAs to generate mature tRNA 5' ends. Little is known concerning the identity and arrangement of functional groups that constitute the active site of this ribozyme. We have used an RNase P RNA-substrate conjugate that undergoes rapid, accurate, and efficient self-cleavage in vitro to probe, by phosphorothioate modification-interference, functional groups required for catalysis. We identify four phosphate oxygens where substitution by sulfur significantly reduces the catalytic rate (50-200-fold). Interference at one site was partially rescued in the presence of manganese, suggesting a direct involvement in binding divalent metal ion cofactors required for catalysis. All sites are located in conserved sequence and secondary structure, and positioned adjacent to the substrate phosphate in a tertiary structure model of the ribozyme-substrate complex. The spatial arrangement of phosphorothioate-sensitive sites in RNase P RNA was found to resemble the distribution of analogous positions in the secondary and potential tertiary structures of other large catalytic RNAs.     

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.     

The P9.1-P9.2 peripheral extension helps guide folding of the Tetrahymena ribozyme.

We have previously proposed a hierarchical model for the folding mechanism of the Tetrahymena ribozyme that may illustrate general features of the folding pathways of large RNAs. While the role of elements in the conserved catalytic core of this ribozyme during the folding process is beginning to emerge, the participation of non-conserved peripheral extensions in the kinetic folding mechanism has not yet been addressed. We now show that the 3'-terminal P9.1-P9.2 extension of the Tetrahymena ribozyme plays an important role during the folding process and appears to guide formation of the catalytic core.     

Structure-function relationships of two closely related group IC3 intron ribozymes from Azoarcus and Synechococcus pre-tRNA

The two group IC3 pre-tRNA introns from Azoarcus and Synechococcus share very analogous secondary structures. They are small group I ribozymes that possess only two peripheral domains, P2 and P9. However, the 3′-splice site hydrolysis activity of the Synechococcus ribozyme critically depends on P2 whereas that of Azoarcus does not, indicating that the structure–function relationships of the two ribozymes are strikingly different despite their structural resemblance. To identify the element(s) that determines the catalytic properties of these ribozymes, we undertook analyses of chimeric ribozymes prepared by swapping their structural elements. We found that the difference can be attributed to a small number of nucleotides within the conserved core region. Further analysis by employing in vitro selection revealed that a base triple interaction (P4bp3 × J6/7-2) is a critical element for determining activity and suggests the existence of a novel base quintuple involving the base triple P4bp5 × J8/7-5.     

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.     

Core requirements for glmS ribozyme self-cleavage reveal a putative pseudoknot structure

The glmS ribozyme is a self-cleaving RNA catalyst that resides in the 5′-untranslated region of glmS mRNA in certain bacteria. The ribozyme is specifically activated by glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein, and is thus proposed to provide a feedback mechanism of riboswitch regulation. Both phylogenetic and biochemical analyses of the glmS ribozyme have established a highly conserved core sequence and secondary structure required for GlcN6P-dependent self-cleavage. However, the high degree of nucleotide conservation offers few clues regarding the higher-order structural organization of the catalytic core. To further investigate core ribozyme structure, minimal ‘consensus-type’ glmS ribozymes that retain GlcN6P-dependent activity were produced. Mutational analyses of consensus-type glmS ribozymes support a model for core ribozyme folding through a pseudoknot structure formed by the interaction of two highly conserved sequence segments. Moreover, GlcN6P-dependent function is demonstrated for bimolecular constructs in which substrate interaction with the ribozyme is minimally comprised of sequence representing that involved in putative pseudoknot formation. These studies suggest that the glmS ribozyme adopts an intricate multi-strand catalytic core through the formation of a pseudoknot structure, and provide a refined model for further considering GlcN6P interaction and GlcN6P-dependent ribozyme function.     

Evidence for preorganization of the glmS ribozyme ligand binding pocket

We have examined the tertiary structure of the ligand-activated glmS ribozyme by a combination of methods with the aim of evaluating the magnitude of RNA conformational change induced by binding of the cofactor, glucosamine 6-phosphate (GlcN6P). Hydroxyl radical footprinting of a trans-acting ribozyme complex identifies several sites of solvent protection upon incubation of the RNA in Mg(2+)-containing solutions, providing initial evidence of the tertiary fold of the ribozyme. Under these folding conditions and at GlcN6P concentrations that saturate the ligand-induced cleavage reaction, we do not observe changes to this pattern. Cross-linking with short-wave UV light of the complex yielded similar overall results. In addition, ribozyme-substrate complexes cross-linked in the absence of GlcN6P could be gel purified and then activated in the presence of ligand. One of these active cross-linked species links the base immediately 3' of the cleavage site to a highly conserved region of the ribozyme core and could be catalytically activated by ligand. Combined with recent studies that argue that GlcN6P acts as a coenzyme in the reaction, our data point to a riboswitch mechanism in which ligand binds to a prefolded active site pocket and assists in catalysis via a direct participation in the reaction chemistry, the local influence on the geometry of the active site constituents, or a combination of both mechanisms. This mode of action is different from that observed for other riboswitches characterized to date, which act by inducing secondary and tertiary structure changes.     

Mutations at the guanosine-binding site of the Tetrahymena ribozyme also affect site-specific hydrolysis.

Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosine-binding site has been localized to the G264-C311 base pair of the Tetrahymena intron on the basis of analysis of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside) (F. Michel et al. (1989) Nature 342, 391-395). We studied the effect of these mutations (G-U, A-C and A-U replacing G264-C311) in the L-21 ScaI version of the Tetrahymena ribozyme. In this enzymatic system (kcat/Km)G monitors the cleavage step. This kinetic parameter decreased by at least 5 x 10(3) when the G264-C311 base pair was mutated to an A-U pair, while (kcat/Km)2AP increased at least 40-fold. This amounted to an overall switch in specificity of at least 2 x 10(5). The nucleophile specificity (G > 2AP for the G-C and G-U pairs, 2AP > G for the A-U and A-C pairs) was consistent with the proposed hydrogen bond between the nucleotide at position 264 and N1 of the nucleophile. Unexpectedly, the A-U and A-C mutants showed a decrease of an order of magnitude in the rate of ribozyme-catalyzed hydrolysis of RNA, in which H2O or OH- replaces G as the nucleophile, whereas the G-U mutant showed a decrease of only 2-fold. The low hydrolysis rates were not restored by raising the Mg2+ concentration or lowering the temperature. In addition, the mutant ribozymes exhibited a pattern of cleavage by Fe(II)-EDTA indistinguishable from that of the wild type, and the [Mg2+]1/2 for folding of the A-U mutant ribozyme was the same as that of the wild type. Therefore the guanosine-binding site mutations do not appear to have a major effect on RNA folding or stability. Because changing G264 affects the hydrolysis reaction without perturbing the global folding of the RNA, we conclude that the catalytic role of this conserved nucleotide is not limited to guanosine binding.     

Role of a conserved J8/7 X P4 base-triple in the Tetrahymena ribozyme

The Tetrahymena group I intron ribozyme folds into a complex three dimensional structure for performing the self-splicing reaction. Catalysis depends on its core structure comprising two helical domains, P4-P6 and P3-P7. The two domains are joined by three sets of conserved base-triple(s) and other tertiary interactions. We found that the disruption of J8/7 X P4, one such conserved base-triple, causes the catalytic ability to deteriorate without altering the folding rate. This suggests that the base-triple stabilizes the active structure of the ribozyme but plays no significant role in RNA folding. By combining the present and previous results, it can be concluded that three sets of conserved base-triples play distinct roles in the Tetrahymena ribozyme.     

Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme

The glmS ribozyme resides in the 5' untranslated region of glmS mRNA and functions as a catalytic riboswitch that regulates amino sugar metabolism in certain Gram-positive bacteria. The ribozyme catalyzes self-cleavage of the mRNA and ultimately inhibits gene expression in response to binding of glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein. We have used nucleotide analog interference mapping (NAIM) and suppression (NAIS) to investigate backbone and nucleobase functional groups essential for ligand-dependent ribozyme function. NAIM using GlcN6P as ligand identified requisite structural features and potential sites of ligand and/or metal ion interaction, whereas NAIS using glucosamine as ligand analog revealed those sites that orchestrate recognition of ligand phosphate. These studies demonstrate that the ligand-binding site lies in close proximity to the cleavage site in an emerging model of ribozyme structure that supports a role for ligand within the catalytic core.     

Designed structural-rearrangement of an active group I ribozyme

The catalytic core of the Tetrahyemena group I ribozyme consists of two functionally different domains, P4-P6 and P3-P7, that are conjugated via multiple tertiary interactions. The sequence encoding the P3-P7 domain is divided into two fragments in its primary sequence although the two domains are physically separable in the three dimensional (3D-) structure of the ribozyme: The sequence encoding the P4-P6 domain is inserted into that of the P3-P7 domain. An artificial rearrangement was designed and attempted for the primary sequence of the P3-P7 domain on the basis of a 3D-structural model and the biochemical data on the ribozyme. The domain in the primary structure was relocated to form a contiguous region while retaining the 3D-structure of the ribozyme required for self-splicing. The topologically rearranged ribozyme exhibited self-splicing activity.     

Two group I ribozymes with different functions in a nuclear rDNA intron.

DiSSU1, a mobile intron in the nuclear rRNA gene of Didymium iridis, was previously reported to contain two independent catalytic RNA elements. We have found that both catalytic elements, renamed GIR1 and GIR2, are group I ribozymes, but with differing functionality. GIR2 carries out the several reactions associated with self-splicing. GIR1 carries out a hydrolysis reaction at an internal processing site (IPS-1). These conclusions are based on the catalytic properties of RNAs transcribed in vitro. Mutation of the P7 pairing segment of GIR2 abrogated self-splicing, while mutation of P7 in GIR1 abrogated hydrolysis at the IPS-1. Much of the P2 stem and all of the associated loop could be deleted without effect on self-splicing. These results are accounted for by a secondary structure model, in which a long P2 pairing segment brings the 5' splice site to the GIR2 catalytic core. GIR1 is the smallest natural group I ribozyme yet reported and is the first example of a group I ribozyme whose presumptive biological function is hydrolysis. We hypothesize that GIR1-mediated cleavage of the excised intron RNA functions in the generation and expression of the mRNA for the intron-encoded endonuclease I-DirI.