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Group II introns are ribozymes with a complex tertiary architecture that is of great interest as a model for RNA folding. Domain 5 (D5) is a highly conserved region of the intron that is considered one of the most critical structures in the catalytic core. Despite its central importance, the means by which D5 interacts with other core elements is unclear. To obtain a map of potential interaction sites, dimethyl sulfate was used to footprint regions of the intron that are involved in D5 binding. These studies were complemented by measurements of D5 binding to a series of truncated intron derivatives. In this way, the minimal region of the intron required for strong D5 association was defined and the sites most likely to represent thermodynamically significant positions of tertiary contact were identified. These studies show that ground-state D5 binding is mediated by tertiary contacts to specific regions of D1, including a tetraloop receptor and an adjacent three-way junction. In contrast, D2 and D3 are not found to stabilize D5 association. These data highlight the significance of D1-D5 interactions and will facilitate the identification of specific tertiary contacts between them.

Domain 6 (D6) of group II introns contains a bulged adenosine that serves as the branch-site during self-splicing. In addition to this adenosine, other structural features in D6 are likely to contribute to the efficiency of branching. To understand their role in promoting self-splicing, the branch-site and surrounding nucleotides were mutagenized. Detailed kinetic analysis on the self-splicing efficiency of the mutants revealed several interesting features. First, elimination of the branch-site does not preclude efficient splicing, which takes place instead through a hydrolytic first step. Second, pairing of the branch-site does not eliminate branching, particularly if the adenosine is involved in a mispair. Third, the G-U pairs that often surround group II intron branch-points contribute to the efficiency of branching. These results suggest that there is a strong driving force for promoting self-splicing by group II introns, which employ a versatile set of different mechanisms for ensuring that splicing is successful. In addition, the behavior of these mutants indicates that a bulged adenosine per se is not the important determinant for branch-site recognition in group II introns. Rather, the data suggest that the branch-site adenosine is recognized as a flipped base, a conformation that can be promoted by a variety of different substructures in RNA and DNA.

Group II introns are large, self-splicing RNAs and mobile genetic elements that provide good model systems for studies of RNA folding. The structures and mechanistic functions of individual domains are being elucidated, and long-range tertiary interactions between the domains are being identified, thus helping to define the three-dimensional architecture of the intron.

Of the proteins required for pre-mRNA splicing, at least four, the DEAH-box proteins, are closely related due to the presence of a central 'RNA helicase-like' region, and extended homology through a large portion of the protein. A major unresolved question is the function of these proteins. Indirect evidence suggests that several of these proteins are catalysts for important structural rearrangements in the spliceosome. However, the mechanism for the proposed alterations is presently unknown. We present evidence that PRP22, a DEAH-box protein required for mRNA release from the spliceosome, unwinds RNA duplexes in a concentration- and ATP-dependent manner. This demonstrates that PRP22 can modify RNA structure directly. We also show that the PRP22-dependent release of mRNA from the spliceosome is an ATP-dependent process and that recombinant PRP22 is an ATPase. Non-hydrolyzable ATP analogs did not substitute for ATP in the RNA-unwinding reaction, suggesting that ATP hydrolysis is required for this reaction. Specific mutation of a putative ATP phosphate-binding motif in the recombinant protein eliminated the ATPase and RNA-unwinding capacity. Significantly, these data suggest that the DEAH-box proteins act directly on RNA substrates within the spliceosome.

Group II intron ai5 gamma was reconstructed into a multiple-turnover ribozyme that efficiently cleaves small oligonucleotide substrates in-trans. This construct makes it possible to investigate sequence specificity, since second-order rate constants (kcat/K(m), or the specificity constant) can be obtained and compared with values for mutant substrates and with other ribozymes. The ribozyme used in this study consists of intron domains 1 and 3 connected in-cis, together with domain 5 as a separate catalytic cofactor. This ribozyme has mechanistic features similar to the first step of reverse-splicing, in which a lariat intron attacks exogenous RNA and DNA substrates, and it therefore serves as a model for the sequence specificity of group II intron mobility. To quantitatively evaluate the sequence specificity of this ribozyme, the WT kcat/Km value was compared to individual kcat/Km values for a series of mutant substrates and ribozymes containing single base changes, which were designed to create mismatches at varying positions along the two ribozyme-substrate recognition helices. These mismatches had remarkably large effects on the discrimination index (1/relative kcat/K(m)), resulting in values > 10,000 in several cases. The delta delta G++ for mismatches ranged from 2 to 6 kcal/mol depending on the mismatch and its position. The high specificity of the ribozyme is attributable to effects on duplex stabilization (1-3 kcal/mol) and unexpectedly large effects on the chemical step of reaction (0.5-2.5 kcal/mol). In addition, substrate association is accompanied by an energetic penalty that lowers the overall binding energy between ribozyme and substrate, thereby causing the off-rate to be faster than the rate of catalysis and resulting in high specificity for the cleavage of long target sequences (> or = 13 nucleotides).

Group I, group II and spliceosomal introns splice by two sequential transesterification reactions. For both spliceosomal and group II introns, the first-step reaction occurs by nucleophilic attack on the 5' splice junction by the 2' hydroxyl of an internal adenosine, forming a 2'-5' phosphodiester branch in the intron. The second reaction joins the two exons with a 3'-5' phosphodiester bond and releases intron lariat. In vitro, group II introns can self-splice by an efficient alternative pathway in which the first-step reaction occurs by hydrolysis. The resulting linear splicing intermediate participates in normal second-step reactions, forming spliced exon and linear intron RNAs. Here we show that the group II intron first-step hydrolysis reaction occurs in vivo in place of transesterification in the mitochondria of yeast strains containing branch-site mutations. As expected, the mutations block branching, but surprisingly still allow accurate splicing. This hydrolysis pathway may have been a step in the evolution of splicing mechanisms.

The most highly conserved nucleotides in D5, an essential active site component of group II introns, consist of an AGC triad, of which the G is invariant. To understand how this G participates in catalysis, the mechanistic contribution of its functional groups was examined. We observed that the exocyclic amine of G participates in ground state interactions that stabilize D5 binding from the minor groove. In contrast, each major groove heteroatom of the critical G (specifically N7 or O6) is essential for chemistry. Thus, major groove atoms in an RNA helix can participate in catalysis, despite their presumed inaccessibility. N7 or O6 of the critical G could engage in critical tertiary interactions with the rest of the intron or they could, together with phosphate oxygens, serve as a binding site for catalytic metal ions.

A stopped-flow fluorescence spectroscopic assay for RNA folding was used to monitor the association of a multicomponent ribozyme derived from group II intron ai5gamma. In the presence of Mg2+, association of a short fluorescein-labeled oligonucleotide substrate with intron domain 1 (D1) resulted in a unique fluorescein emission enhancement, which reflected ribozyme folding and substrate binding. It was found that substrate binding follows a simple bimolecular encounter model, with k(on) approaching the rate of simple duplex formation. The Kd between substrate and D1 was determined to be 11 nM, which is in close agreement with the Kd obtained through oligonucleotide cleavage assays requiring catalytic domain 5 (D5). Ribozyme variants D13 and D135, which contain D3 and/or D5 attached to D1 in-cis, bound substrate with very similar Kd values, suggesting that D1 can fold independently and contains all residues important for ground-state binding to substrate. Both stopped-flow fluorescence assays and chemical modification footprinting data showed that, in all three ribozymes, Mg2+ was required and sufficient for folding. The rates of substrate association and the fraction of active ribozyme showed similar [Mg2+] dependencies, indicating that folding and substrate binding in these three ribozymes are the result of similar processes involving specific, weakly bound Mg2+ ions. The apparent binding constants for the Mg2+ ions were found to be approximately 70 mM in each case. Together, these data show that D1 is an independent folding unit with respect to substrate binding and that specific Mg2+ ions are required for the formation of a distinct tertiary structure in group II introns.

The 2'-hydroxyl on a specific bulged adenosine is the nucleophile during the first step of splicing by group II introns. To understand the means by which the ribozyme core recognizes this adenosine, it was mutagenized and effects on catalytic activity were quantified. The results indicate that a low level of mutational variability is tolerated at the branch-site of group II introns, with no apparent loss of fidelity. Analyses of mutant and modified nucleotides at the branch-site reveal that adenine is recognized primarily through the N6 amino group and by steric exclusion of functionalities found on other bases. The mutational and single atom effects reported here contrast with those observed during spliceosomal processing, suggesting that there are important differences in adenosine recognition by the two systems.

One of the most common RNA tertiary interactions involves the docking of GNRA hairpin loops into stem-loop structures on other regions of RNA. Domain 5 of the group II intron interacts with Domain 1 through such an interaction, which has been characterized thermodynamically and kinetically for the ai5g intron. Using this system, it was possible to test the morphological tolerances of the GNRA tetraloop involved in tertiary interactions. The data presented herein show that a GNRA tetraloop can still participate in tertiary interaction after being physically cut at any phosphodiester linkage within the loop. The "nicked tetraloop" can be expanded by many nucleotides in either direction and the covalently continuous loop can also be expanded without loss of interaction energy. In the context of the nicked tetraloop, the second nucleotide of the tetraloop sequence can be completely deleted without loss of function. By examining radical alterations in tetraloop structure, this study helps define the minimal sequence and structural requirements of a GNRA motif involved in long-range tertiary interaction. It shows that "tetraloop"-like structures capable of forming tertiary interactions can be imbedded in unexpected contexts, such as internal loops and apparently open structure within RNA. It demonstrates that pentaloops and hexaloops can form the same type of interaction, with almost equal affinity, as a tetraloop. Taken together, these data suggest a more generic term for the GNRA tetraloop-receptor interaction: It is proposed herein that the term "GNRA tetraloop" be replaced by "GNn/RA", where n represents a variable number of nucleotides and / indicates that the loop can be divided and interrupted by other sequences.

Domain 5 is an essential active-site component of group II intron ribozymes. The role of backbone substituents in D5 function was explored through synthesis of a series of derivatives containing deoxynucleotides at each position along the D5 strand. Kinetic screens revealed that eight 2'-hydroxyl groups were likely to be critical for activity of D5. Through two separate methods, including competitive inhibition and direct kinetic analysis, effects on binding and chemistry were distinguished. Depending on their function, important 2'-hydroxyl groups lie on opposite faces of the molecule, defining distinct loci for molecular recognition and catalysis by D5.

Self-splicing group II introns are found in bacteria and in the organellar genes in plants, fungi, and yeast. The mechanism for the first step of splicing is generally believed to involve attack of a specific intronic 2'-hydroxyl group on a phosphodiester linkage at the 5'-splice site, resulting in the formation of a lariat intron species. In this paper, we present kinetic and enzymatic evidence that in vitro there are two distinct pathways for group II intron self-splicing: one involves 2'-OH attack and another involves attack of water or hydroxide. These two pathways occur in parallel under all reaction conditions, although either can dominate in the presence of particular salts or protein cofactors. Both pathways are followed by a successful second step of splicing, and either pathway can be highly efficient. We find that the hydrolytic pathway prevails under physiological ionic conditions, while branching predominates at molar concentrations of ammonium ion. The intron is observed to adopt two major active conformations. In order to quantify their individual reaction rates, we applied a mechanistic model describing biphasic parallel kinetic behavior. Kinetic analysis throughout the investigation reveals that there is no coupling between the unproductive "spliced-exon-reopening" reaction (SER) and hydrolysis during the first step of splicing. Conditions that stimulate branching can promote the SER reaction just as efficiently as conditions that stimulate the hydrolytic pathway. Although there is little evidence that it exists in vivo, a hydrolytic splicing pathway for group II introns has important implications for the translation of intron-encoded proteins and the inhibition of intron migration into new genomic positions.

Background: Group II introns are self-splicing RNAs that have mechanistic similarity to the spliceosome complex involved in messenger RNA splicing in eukaryotes. These autocatalytic molecules can be reconfigured into highly specific, multiple-turnover ribozymes that cleave oligonucleotides in trans. We set out to use a simplified system of this kind to study the mechanism of cleavage., Results: Unlike other catalytic RNA molecules, the group II ribozymes cleave DNA linkages almost as readily as RNA linkages. One ribozyme variant cleaves DNA linkages with an efficiency comparable to that of restriction endonuclease EcoRI. Single deoxynucleotide substitutions in the substrate showed that the ribozymes bind substrate without engaging 2'-hydroxyl groups., Conclusions: The ribose 2'-hydroxyl group at the cleavage site has little role in transition-state stabilization by group II ribozymes. Substrate 2'-hydroxyl groups are not involved in substrate binding, suggesting that only base-pairing is required for substrate recognition.

The number of known motifs for RNA folding and RNA tertiary organization is expanding rapidly as we learn more about the diverse biological functions of RNA. Problems in protein and RNA folding have melded in recent investigations of ribonucleoprotein folding. Theoretical and experimental models are rapidly being developed for the pathways and stabilizing forces involved in RNA folding.

By examining the first step of group II intron splicing in the absence of the second step, we have found that there is an interplay of three distinct reactions at the 5'-splice site: branching, reverse branching, and hydrolytic cleavage. This approach has yielded the first kinetic parameters describing eukaryotic branching and establishes that group II intron catalysis can proceed on a rapid timescale. The efficient reversibility of the first step is due to increased conformational organization in the branched intermediate and it has several important mechanistic implications. Reversibility in the first step requires that the second step of splicing serve as a kinetic trap, thus driving splicing to completion and coordinating the first and second step of splicing. Facile reverse branching also provides the intron with a proofreading mechanism to control the fidelity of 5'-splice site selection and it provides a kinetic basis for the apparent mobility of group II introns.

The self-splicing ai5g group II intron was transformed into a three-part ribozyme that site-specifically cleaves small oligonucleotide substrates with multiple turnover. The ribozyme is composed of intron domain 1 (D1, 425 nucleotides), with catalytically essential domain 5 (D5, 58 nucleotides) provided separately as a reaction cofactor. Together, the D1/D5 complex cleaves small substrates analogous in sequence to the 5'-splice site of the intron. Activity of the ribozyme was studied using a combination of single- and multiple-turnover experiments in which the concentrations of the RNA components were varied in order to probe their individual role in the overall mechanism. Values for kcat, Km, and kcat/Km were the same within experimental error for the two enzymological approaches. These kinetic analyses reveal that the ribozyme utilizes a classic Michaelis-Menten reaction mechanism in which the chemical step of catalysis (kcat = kchem = approximately 0.03 min-1 at full saturation) is rate limiting for the overall reaction. The D1/D5 complex binds tightly to the substrate (Km = 6.3 nM) and specifically recognizes sequences both 5' and 3' to the ribozyme cleavage site. These studies represent the first quantitative analysis of group II recognition and affinity for the 5'-splice site. As observed in previous studies on the role of D5 RNA, D5 binds tightly to the ternary complex (Km = 870 nM). The second-order rate constant for RNA cleavage (kcat/Km = 3.3 x 10(6)) is an order of magnitude slower than that observed for other ribozymes in this mechanistic class, all of which are rate-limited by steps other than chemistry. That kcat equals kchem in this ribozyme is supported by the overall kinetic analysis and by the observation that an Rp phosphorothioate is cleaved approximately 3-fold more slowly than a phosphate at the cleavage site. These studies represent a preliminary examination of stereochemical preference by a group II intron active site in the transition state. The substrate specificity, reaction conditions, and mutational sensitivity of this ribozyme are consistent with a reaction analogous to the first step of group II intron self-splicing, although its stereochemical preference is analogous to a second-step reversal.

There is a phylogenetically conserved G.U pair at the 5'-splice site of group I introns. When this is mutagenized to a G-C pair, splicing of these introns is greatly reduced. We have used a ribozyme derived from the Tetrahymena group I intron to compare the binding and reactivity of oligonucleotides that form either a G.U or a G-C pair at this position. Ribozyme binding of oligonucleotides at 42 degrees C was measured by native gel electrophoresis and equilibrium dialysis. Binding of GGCCCUCC (C(-1)P), which base-pairs with the ribozyme guide sequence to form a G-C at the cleavage site, was 10-fold weaker than the binding of GGCCCUCU (U(-1)P), which maintains the conserved G.U pair at the cleavage site. This is surprising since a terminal G-C enhances the binding between oligonucleotides by 20-fold relative to a terminal G.U. Thermal denaturation studies indicate that C(-1)P and several analogs with deoxy substitutions bind the guide-sequence oligonucleotide, GGAGGGAAA, as strongly as they bind the ribozyme. In contrast, U(-1)P binds 240-fold more strongly to the ribozyme than to GGAGGGAAA, a difference that is decreased by deoxy substitutions. Thus, while U(-1)P binds the ribozyme through a combination of base-pairing and specific 2-OH and other tertiary interactions, C(-1)P may bind by base-pairing alone. The substrate GGCCCUCCAAAAA (C(-1)S) is cleaved 100-fold more slowly than GGCCCUCUAAAAA (U(-1)S) and also has a higher propensity to be cleaved at the wrong nucleotide position.(ABSTRACT TRUNCATED AT 250 WORDS)

Ribozyme kinetics and binding studies of a two-piece group II intron were used to mechanistically characterize a reaction analogous to the first step of RNA splicing. Domain 5 RNA (D5) catalyzes specific hydrolysis of an RNA substrate (exD123) composed of sequences surrounding the 5' exon/intron boundary. Both single- and multiple-turnover kinetic analyses produced similar values of kcat (0.04 and 0.1 min-1, respectively) and Km (270 and 190 nM, respectively) for 5' splice site hydrolysis catalyzed by D5. Base pairing is not believed to stabilize the binding of D5 to exD123, so the low Km values suggest that unusual tertiary interactions provide considerable energetic stabilization to this complex. The strength of D5-exD123 binding was confirmed using a new direct binding assay based on gel filtration chromatography. In this initial application of the assay, which systematically underestimates binding by approximately 3-fold, Kd values were obtained in relative agreement with Km. This agreement, together with agreement between kinetically determined variables, suggests that the reaction is described by a straightforward Michaelis-Menten mechanism and that kcat is the rate of the chemical step. This is supported by the log/linear pH/rate profile for kcat which has a slope = 1 up to pH 6.2, consistent with a form of general base catalysis within this linear range. The shape of the plot suggests that the active site responsible for 5' splice site hydrolysis has a pKa of > or = 7.0.

Ribozymes are an important new class of metalloenzymes that have an unlikely feature: they are made entirely of ribonucleic acid (RNA). Metal ions are essential for efficient chemical catalysis by ribozymes and are often required for the stabilization of ribozyme structure. Most ribozymes catalyze reactions at phosphorus centers through one of two major mechanistic pathways, and reaction has been observed at carbon centers. Creative experiments have revealed the position of metal ions in the active site of two ribozymes. The exploitation of variable metal geometry and reactivity has expanded ribozyme chemistry and has facilitated the application of in vitro selection for the creation of novel ribozymes.

In catalysis by group I introns, the helix (P1) containing the RNA cleavage site must be positioned next to the guanosine binding site. We have identified a conserved adenine in the catalytic core that contributes to the stability of this arrangement and propose that it accepts a hydrogen bond from a specific 2'-OH in P1. Such base-backbone tertiary interactions may be generally important to the organization of RNA tertiary structure.

Shortened forms of the group I intron from Tetrahymena catalyse sequence-specific cleavage of exogenous oligonucleotide substrates. The association between RNA enzyme (ribozyme) and substrate is mediated by pairing between an internal guide sequence on the ribozyme and a complementary sequence on the substrate. RNA substrates and cleavage products associate with a binding energy greater than that of base-pairing by approximately 4 kcal-mol-1 (at 42 degrees C), whereas DNA associates with an energy around that expected for base-pairing. It has been proposed that the difference in binding affinity is due to specific 2'-OH groups on an RNA substrate forming stabilizing tertiary interactions with the core of the ribozyme, or that the RNA.RNA helix formed upon association of an RNA substrate and the ribozyme might be more stable than an RNA.DNA helix of the same sequence. To differentiate between these two models, chimaeric oligonucleotides containing deoxynucleotide residues at successive positions along the chain were synthesized, and their equilibrium binding constants for association with the ribozyme were measured directly by a new gel electrophoresis technique. We report here that most of the extra binding energy can be accounted for by discrete RNA-ribozyme interactions, the 2'-OH group on the sugar residue three nucleotides from the cleavage site contributing the most interaction energy. Thus, in addition to the well documented binding of RNA to RNA by base-pairing, 2'-OH groups within a duplex can also mediate association between RNA molecules.

The complex bis(phenanthrenequinone diimine)(bipyridyl)rhodium(III), Rh(phi)2(bpy)3+, cleaves DNA efficiently in a sequence-neutral fashion upon photoactivation so as to provide a novel, high resolution, chemical photofootpring reagent. Photofootprinting of two crystallographically characterized DNA-binding agents, distamycin, a small natural product which binds to DNA in the minor groove, and the endonuclease EcoRI, which binds in the major groove, gave respectively a 5-7 base pair footprint for the drug at its A6 binding site and a 10-12 base pair footprint for the enzyme centered at its recognition site (5'-GAATTC-3'). Both footprints agree closely with the crystallographic results. The photocleavage reaction can be performed using either a high intensity lamp or, conveniently, a simple transilluminator box, and the photoreaction is not inhibited by moderate concentrations of reagents which are sometimes required for examining interactions of molecules with DNA. When compared with other popular footprinting agents, the rhodium complex shows a number of distinct advantages: sequence-neutrality, high resolution, ability to footprint major as well as minor groove-binding ligands, applicability in the presence of additives such as Mg2+ or glycerol, ease of handling, and a sharply footprinted pattern. Light activated footprinting reactions furthermore offer the possibility of examining DNA-binding interactions with time resolution and within the cell.