RNA folding: models and perspectives Tobin R Sosnick y and Tao Pan z

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1 309 RNA folding: models and perspectives Tobin R Sosnick y and Tao Pan z Intrinsic events during RNA folding include conformational search and metal ion binding. Several experimentally testable models have been proposed to explain how large ribozymes accomplish folding. Future challenges include the validation of these models, and the correlation of experimental results and theoretical simulations. Addresses Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA y Institute for Biophysical Dynamics, University of Chicago, Chicago, IL 60637, USA trsosnic@midway.uchicago.edu z taopan@midway.uchicago.edu This review comes from a themed issue on Nucleic acids Edited by Carl C Correll and David MJ Lilley X/03/$ see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI /S X(03) Abbreviation FRET fluorescence resonance energy transfer Introduction RNA folding studies aim to identify the mechanistic relationship between sequence and the functional, three-dimensional structure. A tertiary RNA structure is generally separated into two levels of organization secondary and tertiary structure. The secondary structure is often independently stable. The tertiary structure is composed of secondary structural motifs that are brought together to form modules, domains and the complete structure. As with protein folding studies, most RNA folding studies are conducted in vitro starting from denatured states. The in vitro folding of small proteins probably approximates their folding in vivo, in spite of the differences in how folding is initiated (renaturation versus synthesis). This resemblance is probably because folding is extremely cooperative and nearly the entire chain must be synthesized before any stable structure can form, so that no appreciable folding occurs while the growing polypeptide is still bound to the ribosome [1,2]. In vitro RNA folding, however, may not directly correlate with in vivo folding. An RNA synthesized during transcription can form stable intermediates that are distinct from those formed during renaturation [3 6].Bycontrast, the starting point of in vitro studies is a fully synthesized RNA purified under denaturing conditions. Folding is then initiated by the addition of metal ions. Because of thereadinessofrnatoformstablemotifs,avarietyof intermediates with distinct structures and life-times form along the folding pathway. Precisely which intermediates populate during folding can depend on the initial conditions from which the RNA enters the folding pathway [7 11,12 ]. Nevertheless, physical chemistry studies of RNA folding provide insight into how an RNA sequence dictates the formation of a functional structure. This review of in vitro folding studies of large ribozymes focuses on current issues in the field, including models of the rate-limiting steps and possible experimental tests of these models. We also discuss the exciting single-molecule studies and the challenges associated with predicting RNA folding pathways. Folding kinetics The folding of large ribozymes can be coarsely separated into early and late events. Early events describe the rapid steps that occur when the RNA first encounters the added metal ions. Early events are generally several orders of magnitude faster than late events, which limit folding to the native structure. When folding is initiated from a solution of moderate to high ionic strength, secondary structures are already formed and hence many of the early events may already have occurred, so that only the late events of folding are observable under these conditions. In this review, we define early events of folding as beginning from a highly expanded state without added metal ions [13 15]. Early events: the formation of compact, intermediate states Upon addition of metal ions, a large RNA very rapidly collapses. Two models have been proposed to explain this rapid compaction (Figure 1). In model 1, the chargeneutralized RNA chain collapses nonspecifically, as defined by the lack of protection from hydroxyl radical attack that occurs at solvent-exposed positions. The Tetrahymena group I ribozyme collapses in less than 10 ms [16 ], but protection from hydroxyl radical attack is only detectable after 500 ms [17]. This collapsed species has been described as the counterpart to the molten globules observed during protein folding [14,16 ]. In model 2, the charge-neutralized RNA chain collapses with the formation of defined subdomains or tertiary

2 310 Nucleic acids Figure 1 Model 1 Model 2 Current Opinion in Structural Biology Models of the early events of RNA folding. Model 1 depicts nonspecific collapse and model 2 envisions the presence of specific RNA structures in the collapsed state. The associated Mg 2þ ions are shown in blue and the specific interactions in the collapsed state are shown in red. structure modules stabilized by specific RNA RNA or RNA metal ion interactions. Both the full-length and the catalytic domain of Bacillus subtilis RNase P RNA rapidly collapse to an intermediate state in less than 1 ms [18,19 ]. The intermediate state has the native amount of secondary structure, as well as some tertiary structure, as determined by the hypochromicity change, the degree of surface burial and the size of the RNA. The extent of hydroxyl radical protection of this intermediate state, however, is unknown. The precise nature of the early events has significant implications for later folding events. Because the early events occur very quickly, often <1 ms, there is little time for the RNA to carry out structural rearrangements. At the end of the early phase, the compact state may contain structures that must be disrupted for folding to proceed. If significant RNA structure has to be disrupted for folding to proceed, the RNA is kinetically trapped. On the other hand, if folding is not limited by the unfolding of existing structure, subsequent folding is trap free. Late events: the transition to the native state Late events constitute the rate-limiting step in tertiary RNA folding. Three models have been proposed to explain the rate-limiting steps observed for different RNAs (Figure 2). Models 1 and 2 represent the steps in trap-free folding, focusing on the two obligatory events in the transition to the native RNA structure: conformational search and specific metal ion binding. Model 3 represents RNAs that fold through kinetic traps. Model 3 has been extensively reviewed previously [20] and will not be discussed further. Here, we will focus on issues related to conformational search and the formation of metal ion binding sites. An experimental method used to establish whether the rate-limiting step in RNA folding is trap free relies on changes in the folding rate upon the addition of the denaturant urea. This analysis is based on the observation that these changes reflect RNA surface area burial (or exposure) during a folding transition [21]. The disruption of a structure in a kinetic trap translates to the exposure of surface area upon going from the trapped state to the limiting transition state. Hence, the acceleration of the folding rate in the presence of urea indicates that the ratelimiting step has kinetic traps. The rate-limiting step for an RNA that folds trap free can change in two different ways upon the addition of urea, depending on the Mg 2þ concentration used to fold the RNA [22]. AtMg 2þ concentrations near the transition midpoint, the addition of urea may decelerate folding, either because urea shifts the equilibrium binding of Mg 2þ to the RNA or because urea hinders structure formation. At higher Mg 2þ concentrations, however, the addition of urea may not change the folding rate at all, because the rate-limiting step involves a negligible change in surface burial. Model 1 proposes that RNA and protein folding may be limited by a similar property, the conformational search for a critical folding nucleus [23,24,25 ]. This model of protein folding is supported by the observation that the larger the average sequence distance between native contacts (contact order), the slower the folding rate [26]. One must keep in mind, however, that this relationship is only applicable to proteins that fold in a two-state manner with the conformational search beginning from an unstructured state. By contrast, the conformational search in RNA folding is largely a process of the assembly and docking of

3 RNA folding Sosnick and Pan 311 Figure 2 Model 1: Conformational search Model 2: Forming metal ion site Model 3: Kinetic trap Current Opinion in Structural Biology Models of the rate-limiting step in RNA folding. Model 1 involves conformational search to obtain a critical folding nucleus. Model 2 involves the formation of a specific metal ion binding site on a prebound metal ion (pink sphere) the exchange of a water ligand (dark blue spheres) for an RNA ligand (light blue shapes). Model 3 involves the disruption of a pre-existing structure (green lines). The transition state is depicted in the bracket. preformed structural modules, such as bulged-g motifs and tetraloops. Hence, contact order calculations for RNA folding must be referenced back to the intermediate state populated before the rate-limiting step, not to the completely unfolded state. The relevant or reduced contact order should be calculated based on the distance between these preformed modules. This quantity requires knowledge of the structure of the intermediate before the ratelimiting barrier. Model 2 is based on the observation that many RNA structures contain specifically bound metal ions. The rate-limiting step represents the consolidation of RNA structure around a particular prebound metal ion, resulting in the complete formation of a specific metalion binding site. The consolidation may be directly related to exchange between a metal ion bound water and an RNA ligand. Three experimental methods may be used to differentiate models 1 and 2. If surmounting the limiting kinetic barrier entails structure formation, as proposed in model 1, the addition of urea should make the formation of RNA contacts less favorable and folding should be slowed at all Mg 2þ concentrations. Such folding behavior should be analogous to the behavior of two-state folding proteins. If folding is limited by the formation of a metal ion binding site, as in model 2, urea addition may slow folding at low Mg 2þ concentrations, but would have little impact on the folding rate at high Mg 2þ concentrations. The second method to distinguish the models involves measuring folding rates in the presence of a series of divalent metal ions (e.g. Mg 2þ,Ca 2þ,Sr 2þ and Ba 2þ ). Because this series of metal ions holds on to its coordinated water with decreasing affinity in a predictable way, model 2 predicts that the rate-limiting step would correlate with the known properties of metal ion hydration [19 ]. The third method is to compare the folding rates of circularly permuted isomers of RNA, provided that these isomers fold through the same pathway. Because circular permutation changes the contact order of the RNA chain, model 1 predicts that many circularly permuted RNA isomers would fold at different rates. Model 2, on the other hand, predicts significant changes in the folding rate only for circularly permuted isomers with breaks at or near the rate-limiting metal ion binding site.

4 312 Nucleic acids The timescale of the rate-limiting step in RNA folding depends on the mechanism used by the particular RNA. Folding of the catalytic domain of B. subtilis RNase P RNA probably requires water ligand exchange on a prebound metal ion, taking approximately 200 ms [22]. Folding of a group II intron has been proposed to be limited by conformational search and takes approximately 30 s [27 ]. Folding of the P4 P6 domain of the Tetrahymena group I ribozyme may also be limited by conformational search, although taking just ms [28,29]. Mutational analysis Mutational analysis has been widely applied in protein folding studies to characterize folding transition states [30]. The energetic effect of the mutation of a residue on the folding activation energy relative to the equilibrium stability, quantified as f f ¼ DDG z f =DDG eq, defines the level of the mutated residue s interaction in the transition state. A f f valueofzerooroneindicatesthatthe residue s influence is either absent or fully realized, respectively. The successful application of this analysis requires that the transition state does not change upon mutation. A necessary although insufficient condition is that the folding and unfolding rates have the same denaturant dependence as before (i.e. chevron arms retain their slope). Mutational analysis should be applicable to trap-free RNA folding by measuring the folding rate as a function of Mg 2þ concentration. In all known cases [19,22,31, 32 ], the RNA chevron rolls over or plateaus at both very high and very low Mg 2þ concentrations, indicating the accumulation of a kinetic intermediate before the rate-limiting step (Figure 3). Here, each observed rate is the product of two components: the fractional population of the kinetic intermediate and the rate of traversing the limiting barrier (k f,k u ). Therefore, when comparing the folding of mutants to the folding of the wild-type RNA, a complete Mg 2þ chevron should be measured for unambiguous interpretation. Figure 3 shows several scenarios in which more than one interpretation is possible from incomplete data sets. For example, a f value of 0 could be due 0 or 100% of the structure formed at the site of the substitution, if the folding of the mutant is measured only at a single Mg 2þ concentration. Thermodynamics of tertiary RNA folding The thermodynamics of tertiary RNA folding is intricately related to the structural details of the RNA and its interactions with hydrated metal ions. Because the RNA/ metal ion relationship has not been fully worked out, the field is yet to reach a consensus on how to quantify the free energy of a tertiary RNA structure [33 35,36 ]. Only a few kinetic studies aim for self-consistency between the thermodynamic and kinetic data, which requires the measurement of unfolding rates. A frequently applied model for Mg 2þ -dependent stability is a cooperative binding, Hill-type analysis, where DG ¼ RT ln ð½nš=½išþ ¼ nrt ln ([Mg 2þ ]/K Mg ). The first term of this equation describes the free energy as a function of the fraction of the native state ([N]) and the last populated intermediate at equilibrium ([I]). The second term of this equation describes the derivation of [N] and [I] from fitting the experimental data, where n is the Hill coefficient related to Mg 2þ binding and K Mg is the Mg 2þ concentration required to fold 50% of the RNA. The Hill-type analysis does not consider the interactions with delocalized cations that do not obey mass action principles. These nonspecific interactions can be extremely large and stabilize intermediate states relative to the unfolded state [36 ]. If most such interactions are already present in the intermediate state populated before the native state, they would exert little effect on the relative stability between this intermediate state and the native state. By using this intermediate state as the thermodynamic reference state, the effect of the delocalized Mg 2þ ions on tertiary RNA stability can be minimal. The importance of the thermodynamic reference state was emphasized by the equilibrium folding of homologous catalytic domains from a mesophilic and a thermophilic RNase P RNA [37 ]. The enhanced stability of the thermophilic ribozyme is primarily derived from a large increase in the amount of structure formed in the final transition. The equilibrium intermediate of the thermophilic RNA is much less structured, so that the amount of structure formed in the final folding transition increases by fourfold compared to the mesophilic RNA. In vitro evolution experiments with the Tetrahymena group I intron also show that enhanced thermostability may be related to an increase in folding cooperativity [38 ]. These results suggest that folding cooperativity and stability are intimately related. Single-molecule studies Single-molecule studies have come to RNA folding with a flourish. These studies generally come in two flavors, either observation using fluorescence resonance energy transfer (FRET) of equilibrium fluctuations [11,32,39, 40,41 ] and folding induced by Mg 2þ injection [39], or force-induced unfolding measurements using optical trapping methods [42 ]. Recently, fluorescence correlation spectroscopy and FRET were combined to push single-molecule methods into the submillisecond time regime [32 ]. Single-molecule methods are most valuable when elucidating complex folding pathways. In one case, the folding heterogeneity of the hairpin ribozyme, which had escaped detection in ensemble measurements, was identified and characterized [41 ]. The folding of this ribozyme only involves the docking of two preformed helical motifs

5 RNA folding Sosnick and Pan 313 Figure 3 (a) (b) (c) Nothing changes n 1 and n 2 switch lnk obs K 1 doubles Fraction folded K 2 doubles ln[mg 2+ ] k f doubles k u doubles Current Opinion in Structural Biology Mg 2þ chevron analysis of RNA folding kinetics. (a) AMg 2þ chevron represents the dependence of the reaction rate, k obs,onmg 2þ concentration. A typical Mg 2þ RNA chevron (top panel) rolls over on both sides, indicating the formation of populated kinetic intermediates before the rate-limiting step. The vertex of the chevron is close to the midpoint of the equilibrium folding transition, K Mg (bottom panel). The folding side is at higher Mg 2þ concentrations than K Mg, whereas the unfolding side is at lower Mg 2þ concentrations than K Mg. This type of chevron is described by I eq $ I 1 k $ I 2 k $ N, where I eq is the equilibrium intermediate, and I 1 k and I 2 k are the kinetic intermediates before and after the rate-limiting step, respectively. (b) Scenarios in the analysis of RNA mutations. The wild-type chevron is shown in black and the mutant chevron is shown in red. Top panel: the chevrons are identical, indicating that the interaction involving the mutation is already formed in I eq. Second and third panels: the chevrons shift on the folding or unfolding side, indicating that the particular interaction involving the mutation is formed in the I eq to I 1 k (change in K 1 ¼ Mg 2þ midpoint) or I 2 k to N (change in K 2 ¼ Mg 2þ midpoint) transition, respectively. Fourth and fifth panels: the chevrons have different k f or k u, suggesting that the particular interaction involving the mutation is associated with the rate-limiting step. (c) A case of shifting the transition state involving chevrons with different Hill coefficients, n 1 and n 2. connected by a single phosphodiester bond. Folding of a three-way helical junction derived from the 16S rrna oscillates between an open state and the closed, native state [32 ]. The FRET pair used in both systems should be sensitive to the specific or nonspecific coalescence of the helical motifs (Figure 1). Both studies demonstrate that, at least for these tertiary RNA structures, the collapse of the helical motifs can only occur upon the formation of specific tertiary interactions between the motifs. An important benefit of single-molecule studies is that they can be conducted under equilibrium conditions. Multiple folding and unfolding events can be observed without adjusting solvent conditions. This feature enables direct measurements of folding and unfolding rates (k f,k u ), and the ratio of the intermediates from a single time trajectory. By contrast, the observed relaxation rate in ensemble measurements is the sum of the forward and backward rates, which generally requires the experiments to be conducted under a multitude of conditions to separately obtain k f and k u. The benefit of force-induced unfolding measurements is that the folding reaction coordinate is the well-defined extension of the RNA [42 ]. This feature should aid theoretical calculations of RNA unfolding. Additionally,

6 314 Nucleic acids the height and the location of the rate-limiting barrier can be obtained. These benefits come at a price, however, as the pathway investigated by forced unfolding may not directly relate to folding pathways investigated under equilibrium conditions and folding may not be reversible. Future challenges Because an ultimate goal of RNA folding studies is to predict the stability and folding pathway, theoretical simulations of RNA folding have to be applied. Atomic-level calculations of RNA folding are still at a very early stage. The net stability of RNA represents the small difference between the interactions present in the native state and those present in the last populated intermediate. This daunting task requires dealing with electrostatic interactions with a precision of just a few kcal per mol for the entire system, including the RNA structure, the specific and diffusely bound metal ions, and solvent molecules. The amount of energy required to replace a single inner-sphere water from a bound Mg 2þ can be up to tens of kcal per mol, which illustrates the challenge involved in such calculations. Force-fields commonly used in molecular dynamics simulations of proteins, in spite of years of refinement, still produce substantially disparate results for non-native states [43,44]. This disparity may reflect the fact that the force-fields are optimized for folded structures. There is no a priori reason to expect such force-fields to accurately describe the dynamic features, such as kinetic barriers, of partially folded states. These issues are pertinent to RNA simulations as well. Furthermore, the forcefields have generally been optimized for protein, not for RNA, so that additional modifications are required. Although an accurate, atomic-level description of the folding pathway of a large RNA remains remote, simulations of the folding of small RNA motifs are being performed [45 ]. The energetics of diffuse and specifically bound metal ions for prefolded RNA structures are calculated using the nonlinear Poisson Boltzmann formalism [36,46,47] and Brownian dynamics simulations [48,49] have revealed insightful features. For a folding simulation, these computationally intense calculations must be conducted at each folding step. A more modest goal would be to calculate the stability of the native state relative to the last populated intermediate or the thermodynamic reference state. Although this calculation requires detailed structural knowledge of the intermediate, only the subset of interactions that form in the final transition needs be considered to compute the stability of the native structure. Update A recent single-molecule study of the entire Tetrahymena group I ribozyme elucidated the force-induced unfolding pathway and reiterated the Mg 2þ dependence of kinetic barriers [50 ]. Another single-molecule study investigated the nature of the surprisingly slow docking of the P1 helix onto the prefolded catalytic core of a group I ribozyme [51 ]. Ensemble studies on the same ribozyme mapped the early steps and the degree to which the preferred pathways were dependent upon the initial reaction conditions [52], whereas results with circularly permuted Tetrahymena [53] and Azoarcus group I ribozymes emphasized the importance of topology and folding rates [54 ]. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. De Prat Gay G, Ruiz-Sanz J, Neira JL, Itzhaki LS, Fersht AR: Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module. Proc Natl Acad Sci USA 1995, 92: Kolb VA, Makeyev EV, Spirin AS: Co-translational folding of an eukaryotic multidomain protein in a prokaryotic translation system. J Biol Chem 2000, 275: Pan T, Artsimovitch I, Fang X, Landick R, Sosnick TR: Folding of a large ribozyme during transcription and the effect of the elongation factor NusA. Proc Natl Acad Sci USA 1999, 96: Nikolcheva T, Woodson SA: Facilitation of group I splicing in vivo: misfolding of the Tetrahymena IVS and the role of ribosomal RNA exons. J Mol Biol 1999, 292: Waldsich C, Masquida B, Westhof E, Schroeder R: Monitoring intermediate folding states of the td group I intron in vivo. EMBO J 2002, 21: Diegelman-Parente A, Bevilacqua PC: A mechanistic framework for co-transcriptional folding of the HDV genomic ribozyme in the presence of downstream sequence. J Mol Biol 2002, 324: Pan T, Fang X, Sosnick TR: Pathway modulation, circular permutation and rapid RNA folding under kinetic control. J Mol Biol 1999, 286: Pan J, Deras ML, Woodson SA: Fast folding of a ribozyme by stabilizing core interactions: evidence for multiple folding pathways in RNA. J Mol Biol 2000, 296: Russell R, Herschlag D: Probing the folding landscape of the Tetrahymena ribozyme: commitment to form the native conformation is late in the folding pathway. J Mol Biol 2001, 308: Heilman-Miller SL, Pan J, Thirumalai D, Woodson SA: Role of counterion condensation in folding of the Tetrahymena ribozyme. II. Counterion-dependence of folding kinetics. J Mol Biol 2001, 309: Russell R, Zhuang X, Babcock HP, Millett IS, Doniach S, Chu S, Herschlag D: Exploring the folding landscape of a structured RNA. Proc Natl Acad Sci USA 2002, 99: Takamoto K, He Q, Morris S, Chance MR, Brenowitz M: Monovalent cations mediate formation of native tertiary structure of the Tetrahymena thermophila ribozyme. 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