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.     

Role of counterion condensation in folding of the Tetrahymena ribozyme. II. Counterion-dependence of folding kinetics

Condensed counterions contribute to the stability of compact structures in RNA, largely by reducing electrostatic repulsion among phosphate groups. Varieties of cations induce a collapsed state in the Tetrahymena ribozyme that is readily transformed to the catalytically active structure in the presence of Mg2+. Native gel electrophoresis was used to compare the effects of the valence and size of the counterion on the kinetics of this transition. The rate of folding was found to decrease with the charge of the counterion. Transitions in monovalent ions occur 20- to 40-fold faster than transitions induced by multivalent metal ions. These results suggest that multivalent cations yield stable compact structures, which are slower to reorganize to the native conformation than those induced by monovalent ions. The folding kinetics are 12-fold faster in the presence of spermidine3+ than [Co(NH3)6]3+, consistent with less effective stabilization of long-range RNA interactions by polyamines. Under most conditions, the observed folding rate decreases with increasing counterion concentration. In saturating amounts of counterion, folding is accelerated by addition of urea. These observations indicate that reorganization of compact intermediates involves partial unfolding of the RNA. We find that folding of the ribozyme is most efficient in a mixture of monovalent salt and Mg2+. This is attributed to competition among counterions for binding to the RNA. The counterion dependence of the folding kinetics is discussed in terms of the ability of condensed ions to stabilize compact structures in RNA.     

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.     

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.     

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.     

Role of counterion condensation in folding of the Tetrahymena ribozyme. I. Equilibrium stabilization by cations

Folding of RNA into an ordered, compact structure requires substantial neutralization of the negatively charged backbone by positively charged counterions. Using a native gel electrophoresis assay, we have examined the effects of counterion condensation upon the equilibrium folding of the Tetrahymena ribozyme. Incubation of the ribozyme in the presence of mono-, di- and trivalent ions induces a conformational state that is capable of rapidly forming the native structure upon brief exposure to Mg2+. The cation concentration dependence of this transition is directly correlated with the charge of the counterion used to induce folding. Substrate cleavage assays confirm the rapid onset of catalytic activity under these conditions. These results are discussed in terms of classical counterion condensation theory. A model for folding is proposed which predicts effects of charge, ionic radius and temperature on counterion-induced RNA folding transitions.     

The fastest global events in RNA folding: electrostatic relaxation and tertiary collapse of the Tetrahymena ribozyme

Large RNAs can collapse into compact conformations well before the stable formation of the tertiary contacts that define their final folds. This study identifies likely physical mechanisms driving these early compaction events in RNA folding. We have employed time-resolved small-angle X-ray scattering to monitor the fastest global shape changes of the Tetrahymena ribozyme under different ionic conditions and with RNA mutations that remove long-range tertiary contacts. A partial collapse in each of the folding time-courses occurs within tens of milliseconds with either monovalent or divalent cations. Combined with comparison to predictions from structural models, this observation suggests a relaxation of the RNA to a more compact but denatured conformational ensemble in response to enhanced electrostatic screening at higher ionic concentrations. Further, the results provide evidence against counterion-correlation-mediated attraction between RNA double helices, a recently proposed model for early collapse. A previous study revealed a second 100 ms phase of collapse to a globular state. Surprisingly, we find that progression to this second early folding intermediate requires RNA sequence motifs that eventually mediate native long-range tertiary interactions, even though these regions of the RNA were observed to be solvent-accessible in previous footprinting studies under similar conditions. These results help delineate an analogy between the early conformational changes in RNA folding and the "burst phase" changes and molten globule formation in protein folding.     

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.     

Linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of the Tetrahymena ribozyme

We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of Tetrahymena thermophila ribozyme by examining the Mg2+-induced folding and the urea-induced denaturation of the folded state as a function of Na+ under equilibrium folding conditions using hydroxyl radical footprinting. These studies allowed a thermodynamic examination of eight discrete protection sites within P4-P6 that are involved in several tertiary structure contacts. Monovalent ions compete with Mg2+ ions in mediating P4-P6 folding. The urea denaturation isotherms demonstrated DeltaDeltaG values of >2 kcal x mol(-1) in experiments conducted in 10 versus 200 mM NaCl at a constant 10 mM MgCl2. However, the individual-site isotherms reported by footprinting revealed that larger than average changes in DeltaG values were localized to specific sites within the Mg2+-rich A-bulge. The competitive effects of monovalent ions were less when K+ rather than Na+ was the monovalent cation present. This result indicates the importance of the specific K+ binding sites that are associated with AA-platform structures to P4-P6 folding and stability. These site-specific footprinting data provide quantitative and site-specific measurements of the ion-linked stability for P4-P6 that are interpreted with respect to crystallographic data.     

RNA folding and misfolding of the hammerhead ribozyme

The hammerhead ribozyme undergoes a well-defined two-stage folding process induced by the sequential binding of two magnesium ions. These probably correspond to the formation of domain 2 (0-500 microM magnesium ions) and domain 1 (1-20 mM magnesium ions), respectively. In this study we have used fluorescence resonance energy transfer (FRET) to analyze the ion-induced folding of a number of variants of the hammerhead ribozyme. We find that both A14G and G8U mutations are highly destabilizing, such that these species are essentially unfolded under all conditions. Thus they appear to be blocked in the first stage of the folding process, and using uranyl-induced photocleavage we show that the core is completely accessible to this probe under these conditions. Changes at G5 do not affect the first transition but appear to provide a blockage at the second stage of folding; this is true of changes in the sugar (removal of the 2'-hydroxyl group) and base (G5C mutation, previously studied by comparative gel electrophoresis). Arrest of folding at this intermediate stage leads to a pattern of uranyl-induced photocleavage that is changed from the wild-type, but suggests a structure less open than the A14G mutant. Specific photocleavage at G5 is found only in the wild-type sequence, suggesting that this ion-binding site is formed late in the folding process. In addition to folding that is blocked at selected stages, we have also observed misfolding. Thus the A13G mutation appears to result in the ion-induced formation of a novel tertiary structure.     

Template-directed primer extension catalyzed by the Tetrahymena ribozyme.

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

Modulating the splicing activity of Tetrahymena ribozyme via RNA self-assembly

The internal guiding sequence (IGS) is normally located at the 5' end of trans-splicing ribozymes that are derived from the Tetrahymena group I intron, and is required for the recognition of substrate RNAs and for trans-splicing reactions. Here, we separated the Tetrahymena group I intron at the L2 loop to produce two fragments: the IGS-containing substrate, and the IGS-lacking ribozyme. We show here that two fragments can complex not through the IGS interaction but under the guidance of appended interacting nucleotides, and perform trans-splicing. The splicing reactions took place both in vitro and in mammalian cells, and the spliced mRNA product from the self-assembled ribozyme complex can be translated into functional proteins in vivo. The splicing efficiency was dependent on the length of appending nucleotides.     

Metal ions in ribozyme folding and catalysis

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

Evolution of Tetrahymena ribozyme mutants with increased structural stability

Determining how large RNA molecules stabilize their tertiary structures is critical for understanding how they perform their biological functions. Here we use in vitro selection to identify active variants of the Tetrahymena ribozyme with increased stability. The mutant pool converged to a single family that shared nine mutations; an RNA representing the consensus sequence was structurally more stable by 10.5 degrees C and catalytically active at elevated temperatures. Remarkably, of the nine altered sites, most are already known to be involved in tertiary interactions, and the stabilizing mutations primarily improve the packing interactions in the molecular interior. The wild type ribozyme and the selected mutants provide pairs of mesophilic and thermophilic homologs for studying the origin of their thermal stability.     

Probing the Tetrahymena group I ribozyme reaction in both directions

The Tetrahymena L-21 ScaI ribozyme derived from the self-splicing group I intron catalyzes a reversible reaction analogous to the first step of self-splicing: CCCUCUA (S) + [UC]G right harpoon over left harpoon CCCUCU (P) + [UC]GA. To relate our understanding of the ribozyme to the self-splicing reaction and to further the mechanistic dissection of the ribozyme reaction, we have established a quantitative kinetic and thermodynamic framework for the forward and reverse reaction of the L-21 ScaI ribozyme under identical conditions. Examination of the framework shows that binding of products is cooperative with binding enhanced 5-fold, as was observed previously for binding of the substrates. Further, binding of UCGA is 12-fold weaker than binding of the unphosphorylated UCG, analogous to the 20-fold weaker binding of phosphorylated S relative to P; the molecular interactions underlying the stronger binding of UCG were traced to the 3'-hydroxyl group of UCG. The symmetrical effects on binding of substrates and products result in the equilibrium between ribozyme-bound species, K(int), that is essentially unperturbed from the solution equilibrium, K(ext) (K(int) = [E.P.UCGA]/[E.S.UCG] = 4.6 and K(ext) = [P][UCGA]/[S][UCG] = 1.9). Last, we show that the pK(a) values of the nucleophiles in the forward and reverse reactions are >/=10. This observation suggests that metal ion activation of the nucleophile and stabilization of the leaving group can only account for a portion of the rate enhancement of this ribozyme. These and prior results suggest that the Tetrahymena group I ribozyme, analogous to protein enzymes, uses multiple catalytic strategies to achieve its large rate enhancement.