Two major tertiary folding transitions of the Tetrahymena catalytic RNA.

The L-21 Tetrahymena ribozyme, an RNA molecule with sequence-specific endoribonuclease activity derived from a self-splicing group I intron, provides a model system for studying the RNA folding problem. A 160 nucleotide, independently folding domain of tertiary structure (the P4-P6 domain) comprises about half of the ribozyme. We now apply Fe(II)-EDTA cleavage to mutants of the ribozyme to explore the role of individual structural elements in tertiary folding of the RNA at equilibrium. Deletion of peripheral elements near the 3' end of the ribozyme destabilizes a region of the catalytic core (P3-P7) without altering the folding of the P4-P6 domain. Three different mutations within the P4-P6 domain that destabilize its folding also shift the folding of the P3-P7 region of the catalytic core to higher MgCl2 concentrations. We conclude that the role of the extended P4-P6 domain and of the 3'-terminal peripheral elements is at least in part to stabilize the catalytic core. The organization of RNA into independently folding domains of tertiary structure may be common in large RNAs, including ribosomal RNAs. Furthermore, the observation of domain-domain interactions in a catalytic RNA supports the feasibility of a primitive spliceosome without any proteins.     

Perturbation of the hierarchical folding of a large RNA by the destabilization of its Scaffold's tertiary structure

The P4-P6 domain serves as a scaffold against which the periphery and catalytic core organize and fold during Mg2+-mediated folding of the Tetrahymena thermophila ribozyme. The most prominent structural motif of the P4-P6 domain is the tetraloop-tetraloop receptor interaction which "clamps" the distal parts of its hairpin-like structure. Destabilization of the tertiary structure of the P4-P6 domain by perturbation of the tetraloop-tetraloop receptor interaction alters the Mg2+-mediated folding pathway. The folding hierarchy of P5c approximately P4-P6 > periphery > catalytic core that is a striking attribute of the folding of the wild-type RNA is abolished. The initial steps in folding of the mutant RNA are > or =50-fold faster than those of the wild-type ribozyme with the earliest observed tertiary contacts forming around regions known to specifically bind Mg2+. The interaction between the mutant tetraloop and the tetraloop receptor appears coincidently with slowly forming catalytic core tertiary contacts. Thus, the stability conferred upon the P4-P6 domain by the tetraloop-tetraloop receptor interaction dictates the preferred folding pathway by stabilizing an early intermediate. A sub-denaturing concentration of urea diminishes the early barrier to folding the wild-type ribozyme along with complex effects on the subsequent steps of folding the wild-type and mutant RNA.     

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.     

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.     

Fast formation of the P3-P7 pseudoknot: a strategy for efficient folding of the catalytically active ribozyme

Formation of the P3-P7 pseudoknot structure, the core of group I ribozymes, requires long-range base pairing. Study of the Tetrahymena ribozyme appreciates the hierarchical folding of the large, multidomain RNA, in which the P3-P7 core folds significantly slower than do the other domains. Here we explored the formation of the P3-P7 pseudoknot of the Candida ribozyme that has been reported to concertedly fold to the catalytically active structure with a rate constant of 2 min(-1). We demonstrate that pseudoknot formation occurs during the rapid ribozyme compaction, coincident with formation of many tertiary interactions of the ribozyme. A low physiological concentration of magnesium (1.5 mM) is sufficient to fully support the pseudoknot formation. The presence of nonnative intermediates containing an unfolded P3-P7 region is evident. However, catalysis-based analysis shows these nonnative intermediates are stable and fail to convert to the catalytically active structure, suggesting that rapid pseudoknot formation is essential for folding of the active ribozyme. Interestingly, RNAstructure predicts no stable Alt P3 structure for the Candida ribozyme, but two stable Alt P3s for the Tetrahymena ribozyme, explaining the dramatic difference in folding of the P3-P7 core of these two ribozymes. We propose that rapid formation of the P3-P7 pseudoknot represents a folding strategy ensuring efficient production of the catalytically active structure of group I ribozymes, which sheds new light on the mechanism of effective ribozyme folding in vivo.     

Communication between RNA folding domains revealed by folding of circularly permuted ribozymes

To study the role of sequence and topology in RNA folding, we determined the kinetic folding pathways of two circularly permuted variants of the Tetrahymena group I ribozyme, using time-resolved hydroxyl radical footprinting. Circular permutation changes the distance between interacting residues in the primary sequence, without changing the native structure of the RNA. In the natural ribozyme, tertiary interactions in the P4-P6 domain form in 1 s, while interactions in the P3-P9 form in 1-3 min at 42 degrees C. Permutation of the 5' end to G111 in the P4 helix allowed the stable P4-P6 domain to fold in 200 ms at 30 degrees C, five times faster than in the wild-type RNA, while the other domains folded five times more slowly (5-8 min). By contrast, circular permutation of the 5' end to G303 in J8/7 decreased the folding rate of the P4-P6 domain. In this permuted RNA, regions joining P2, P3 and P4 were protected in 500 ms, while the P3-P9 domain was 60-80% folded within 30 s. RNase T(1) digestion and FMN photocleavage showed that circular permutation of the RNA sequence alters the initial ensemble of secondary structures, thereby changing the tertiary folding pathways. Our results show that the natural 5'-to-3' order of the structural domains in group I ribozymes optimizes structural communication between tertiary domains and promotes self-assembly of the catalytic center.     

Multiple monovalent ion-dependent pathways for the folding of the L-21 Tetrahymena thermophila ribozyme

Synchrotron hydroxyl radical (*OH) footprinting is a technique that monitors the local changes in solvent accessibility of the RNA backbone on milliseconds to minutes time-scales. The Mg(2+)-dependent folding of the L-21 Sca 1 Tetrahymena thermophila ribozyme has been followed using this technique at an elevated concentration of monovalent ion (200 mM NaCl) and as a function of the initial annealing conditions and substrate. Previous studies conducted at low concentrations of monovalent ion displayed sequential folding of the P4-P6 domain, the peripheral helices and the catalytic core, with each protection displaying monophasic kinetics. For ribozyme annealed in buffer containing 200 mM NaCl and folded by the addition of 10 mM MgCl(2), multiple kinetic phases are observed for *OH protections throughout the ribozyme. The independently folding P4-P6 domain is the first to fold with its protections displaying 50-90% burst phase amplitudes. That the folding of P4-P6 within the ribozyme does not display the 100% burst phase of isolated P4-P6 at 200 mM NaCl shows that interactions with the remainder of the ribozyme impede this domain's folding. In addition, *OH protections constituting each side of a tertiary contact are not coincident in some cases, consistent with the formation of transient non-native interactions. While the peripheral contacts and triple helical scaffold exhibit substantial burst phases, the slowest protection to appear is J8/7 in the catalytic core, which displays a minimal burst amplitude and whose formation is coincident with the recovery of catalytic activity. The number of kinetic phases as well as their amplitudes and rates are different when the ribozyme is annealed in low-salt buffer and folded by the concomitant addition of monovalent and divalent cations. Annealed substrate changes the partitioning of the ribozyme among the multiple folding populations. These results provide a map of the early steps in the ribozyme's folding landscape and the degree to which the preferred pathways are dependent upon the initial reaction conditions.     

Perturbed folding kinetics of circularly permuted RNAs with altered topology

The folding pathway of the Tetrahymena ribozyme correlates inversely with the sequence distance between native interactions, or contact order. The rapidly folding P4-P6 domain has a low contact order, while the slowly folding P3-P7 region has a high contact order. To examine the role of topology and contact order in RNA folding, we screened for circular permutants of the ribozyme that retain catalytic activity. Permutants beginning in the P4-P6 domain fold 5 to 20 times more slowly than the wild-type ribozyme. By contrast, 50% of a permuted RNA that disjoins a non-native interaction in P3 folds tenfold faster than the wild-type ribozyme. Hence, the probability of rapidly folding to the native state depends on the topology of tertiary domains.     

Monovalent ion-mediated folding of the Tetrahymena thermophila ribozyme

The time-course of monovalent cation-induced folding of the L-21 Sca1 Tetrahymena thermophila ribozyme and a selected mutant was quantitatively followed using synchrotron X-ray (.OH) footprinting. Initiating folding by increasing the concentration of either Na+ or K+ to 1.5M from an initial condition of approximately 0.008 M Na+ at 42 degrees C resulted in the complete formation of tertiary contacts within the P5abc subdomain and between the peripheral helices within the dead time of our measurements (k>50 s(-1)). These results contrast with folding rates of 2-0.2 s(-1) previously observed for formation of these contacts in 10mM Mg2+ from the same initial condition. Thus, the initial formation of native tertiary contacts is inhibited by divalent but not monovalent cations. The native contacts within the catalytic core form without a detectable burst phase at rates of 0.4-1.0 s(-1) in a manner reminiscent of the Mg2+-dependent folding behavior, although tenfold faster. The tertiary interactions stabilizing the catalytic core interaction with P4-P6 and P2.1, as well as one of the protections internal for the P4-P6 domain, display progress curves with appreciable burst amplitudes and a phase comparable in rate to that of the catalytic core. That the slow folding of the ribozyme's core is a consequence of the alt-P3 secondary structure is shown by the 100% burst phase amplitudes that are observed for folding of the U273A mutant ribozyme within which the native secondary structure (P3) is strengthened. Thus, formation of a misfolded intermediate(s) resulting from the alt-P3 secondary structure is independent of ion valency while the rate at which the respective intermediates are resolved is sensitive to ion valency. The overall portrait painted by these results is that ion valency differentially affects steps in the folding process and that folding in monovalent ion alone for the U273A mutant Tetrahymena ribozyme is fast and direct.     

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.     

Monitoring intermediate folding states of the td group I intron in vivo

Group I introns consist of two major structural domains, the P4-P6 and P3-P9 domains, which assemble through interactions with peripheral extensions to fold into an active ribozyme. To assess group I intron folding in vivo, we probed the structure of td wild-type and mutant introns using dimethyl sulfate. The results suggest that the majority of the intron population is in the native state in accordance with the current structural model, which was refined to include two novel tertiary contacts. The importance of the loop E motif in the P7.1-P7.2 extension in assisting ribozyme folding was deduced from modeling and mutational analyses. Destabilization of stem P6 results in a deficiency in tertiary structure formation in both major domains, while weakening of stem P7 only interferes with folding of the P3-P9 domain. The different impact of mutations on the tertiary structure suggests that they interfere with folding at different stages. These results provide a first insight into the structure of folding intermediates and suggest a putative order of events in a hierarchical folding pathway in vivo.     

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.     

A Tyrosyl-tRNA Synthetase Suppresses Structural Defects in the Two Major Helical Domains of the Group I Intron Catalytic Core

TheNeurospora crassamitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain). To investigate how CYT-18 stabilizes the active RNA structure, we used anEscherichia coligenetic assay based on the phage T4tdintron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long- range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain. Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees. The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in thetdintron. Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site.     

Fast folding of a ribozyme by stabilizing core interactions: evidence for multiple folding pathways in RNA

Folding of the Tetrahymena ribozyme under physiological conditions in vitro is limited by slow conversion of long-lived intermediates to the active structure. These intermediates arise because the most stable domain of the ribozyme folds 10-50 times more rapidly than the core region containing helix P3. Native gel electrophoresis and time-resolved X-ray-dependent hydroxyl radical cleavage revealed that mutations that weaken peripheral interactions between domains accelerated folding fivefold, while a point mutation that stabilizes P3 enabled 80 % of the mutant RNA to reach the native conformation within 30 seconds at 22 degrees C. The P3 mutation increased the folding rate of the catalytic core as much as 50-fold, so that both domains of the ribozyme were formed at approximately the same rate. The results show that the ribozyme folds rapidly without significantly populating metastable intermediates when native interactions in the ribozyme core are stabilized relative to peripheral structural elements.     

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.     

Kinetic pathway for folding of the Tetrahymena ribozyme revealed by three UV-inducible crosslinks.

The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures.     

Pathway modulation, circular permutation and rapid RNA folding under kinetic control

The thermodynamics and folding kinetics of a circularly permuted construct of the ribozyme from Bacillus subtilis RNase P are analyzed and compared with the folding properties of the wild-type ribozyme using optical spectroscopy and catalytic activity. The folding of the wild-type ribozyme is slow due to the rearrangement of kinetically trapped species containing misfolded structures. To test whether any misfolded structure arises from interactions between the two independently folding domains of the RNase P RNA, a circular permuted form was created where one of the two phosphodiester bonds connecting these domains is broken. This construct folds approximately 15-fold faster (t1/2 approximately nine seconds) than the wild-type ribozyme at 37 degreesC. While the complete folding of both domains is kinetically indistinguishable in the wild-type ribozyme, one domain folds much faster than the other domain in the circularly permuted construct. Hence, the major kinetic trap in the folding of the wild-type RNase P RNA involves interdomain interactions. This kinetic trap is avoidable at 37 degreesC in the circularly permuted RNA. However, at temperatures below 30 degreesC or when refolding begins from an equilibrium intermediate stabilized by submillimolar concentrations of Mg2+, a subpopulation containing an interdomain misfold still forms. These results indicate that the folding pathway of this large RNA is highly malleable and can be under kinetic control.     

Deletion of the P5abc peripheral element accelerates early and late folding steps of the Tetrahymena group I ribozyme

The P5abc peripheral element stabilizes the Tetrahymena group I ribozyme and enhances its catalytic activity. Despite its beneficial effects on the native structure, prior studies have shown that early formation of P5abc structure during folding can slow later folding steps. Here we use a P5abc deletion variant E(deltaP5abc) to systematically probe the role of P5abc throughout tertiary folding. Time-resolved hydroxyl radical footprinting shows that E(deltaP5abc) forms its earliest stable tertiary structure on the millisecond time scale, approximately 5-fold faster than the wild-type ribozyme, and stable structure spreads throughout E(deltaP5abc) in seconds. Nevertheless, activity measurements show that the earliest detectable formation of native E(deltaP5abc) ribozyme is much slower (approximately 0.6 min(-1)), in a manner similar to that of the wild type. Also similar, only a small fraction of E(deltaP5abc) attains the native state on this time scale under standard conditions at 25 degrees C, whereas the remainder misfolds; footprinting experiments show that the misfolded conformer shares structural features with the long-lived misfolded conformer of the wild-type ribozyme. Thus, P5abc does not have a large overall effect on the rate-limiting step(s) along this pathway. However, once misfolded, E(deltaP5abc) refolds to the native state 80-fold faster than the wild-type ribozyme and is less accelerated by urea, indicating that P5abc stabilizes the misfolded structure relative to the less-ordered transition state for refolding. Together, the results suggest that, under these conditions, even the earliest tertiary folding intermediates of the wild-type ribozyme represent misfolded species and that P5abc is principally a liability during the tertiary folding process.     

Folding mechanism of the Tetrahymena ribozyme P4-P6 domain

Synchrotron X-ray-dependent hydroxyl radical footprinting was used to probe the folding kinetics of the P4-P6 domain of the Tetrahymena group I ribozyme, which forms a stable, closely packed tertiary structure. The 160-nt domain folds independently at a similar rate (approximately 2 s(-1)) as it does in the ribozyme, when folding is measured in 10 mM sodium cacodylate and 10 mM MgCl(2). Surprisingly, tertiary interactions around a three-helix junction (P5abc) within the P4-P6 domain fold at least 25 times more rapidly (k >/= 50 s(-1)) in isolation, than when part of the wild-type P4-P6 RNA. This difference implies that long-range interactions in the P4-P6 domain can interfere with folding of P5abc. P4-P6 was observed to fold much faster at higher ionic strength than in 10 mM sodium cacodylate. Analytical centrifugation was used to measure the sedimentation and diffusion coefficients of the unfolded RNA. The hydrodynamic radius of the RNA decreased from 58 to 46 A over the range of 0-100 mM NaCl. We propose that at low ionic strength, the addition of Mg(2+) causes the domain to collapse to a compact intermediate where P5abc is trapped in a non-native structure. At high ionic strength, the RNA rapidly collapses to the native structure. Faster folding most likely results from a different average initial conformation of the RNA in higher salt conditions.     

The folding of the hairpin ribozyme: dependence on the loops and the junction

In its natural context, the hairpin ribozyme is constructed around a four-way helical junction. This presents the two loops that interact to form the active site on adjacent arms, requiring rotation into an antiparallel structure to bring them into proximity. In the present study we have compared the folding of this form of the ribozyme and subspecies lacking either the loops or the helical junction using fluorescence resonance energy transfer. The complete ribozyme as a four-way junction folds into an antiparallel structure by the cooperative binding of magnesium ions, requiring 20-40 microM for half-maximal extent of folding ([Mg2+]1/2) and a Hill coefficient n = 2. The isolated junction (lacking the loops) also folds into a corresponding antiparallel structure, but does so noncooperatively (n = 1) at a higher magnesium ion concentration ([Mg2+]1/2 = 3 mM). Introduction of a G + 1A mutation into loop A of the ribozyme results in a species with very similar folding to the simple junction, and complete loss of ribozyme activity. Removal of the junction from the ribozyme, replacing it either with a strand break (serving as a hinge) or a GC5 bulge, results in greatly impaired folding, with [Mg2+]1/2 > 20 mM. The results indicate that the natural form of the ribozyme undergoes ion-induced folding by the cooperative formation of an antiparallel junction and loop-loop interaction to generate the active form of the ribozyme. The four-way junction thus provides a scaffold in the natural RNA that facilitates the folding of the ribozyme into the active form.