Monovalent cations mediate formation of native tertiary structure of the Tetrahymena thermophila ribozyme

The formation of individual tertiary contacts of the Tetrahymena L-21 Sca I ribozyme has been monitored by hydroxyl radical footprinting and its global conformation by analytical ultracentrifugation as a function of monovalent ion concentration in the absence of divalent ions. Advanced methods of data analysis, which allow the hydroxyl radical reactivity of every nucleotide to be quantified, permit monitoring of each and every structural element of the RNA. Monovalent ion-mediated global compaction of the ribozyme is accompanied by the formation of native tertiary contacts; most native tertiary contacts are evident except several that are located near where divalent ions are observed in crystallographic structures. Non-native tertiary contacts are also observed at low but not high concentrations of monovalent ions. In light of recent studies that have shown that the presence of monovalent ions greatly accelerates the Mg2+-dependent folding of the Tetrahymena ribozyme, the present studies suggest that Na+ concentration changes not only the starting position of the RNA on its folding funnel but also pushes it deep into the well by forming native tertiary contacts and, thus, favoring fast and correct folding pathways.     

RNA tertiary folding monitored by fluorescence of covalently attached pyrene

The pathways by which large RNAs adopt tertiary structure are just beginning to be explored, and new methods that reveal RNA folding are highly desirable. Here we report an assay for RNA tertiary folding in which the fluorescence of a covalently incorporated chromophore is monitored. Folding of the 160-nucleotide Tetrahymena group I intron P4-P6 domain was used as a test system. Guided by the P4-P6 X-ray crystal structure, we chose a nucleotide (U107) for which derivatization at the 2'-position should not perturb the folded conformation. A 15-mer RNA oligonucleotide with a 2'-amino substitution at U107 was derivatized with a pyrene chromophore on a variable-length tether, and then ligated to the remainder of P4-P6, providing a site-specifically pyrene-labeled P4-P6 derivative. Upon titration of the pyrene-derivatized P4-P6 with Mg(2+), the equilibrium fluorescence intensity reversibly increased several-fold, as expected if the probe's chemical microenvironment changes as the RNA to which it is attached folds. The concentration and specificity of divalent ions required to induce the fluorescence change (Mg(2+) approximately Ca(2+) > Sr(2+)) correlated well with biochemical folding assays that involve nondenaturing gel electrophoresis. Furthermore, mutations in P4-P6 remote from the chromophore that shifted the Mg(2+) folding requirement on nondenaturing gels also affected in a predictable way the Mg(2+) requirement for the fluorescence increase. Initial stopped-flow studies with millisecond time resolution suggest that this fluorescence method will be useful for following the kinetics of P4-P6 tertiary folding. We conclude that a single site-specifically tethered chromophore can report the formation of global structure of a large RNA molecule, allowing one to monitor both the equilibrium progress and the real-time kinetics of RNA tertiary folding.     

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.     

The contribution of metal ions to the structural stability of the large ribosomal subunit

Both monovalent cations and magnesium ions are well known to be essential for the folding and stability of large RNA molecules that form complex and compact structures. In the atomic structure of the large ribosomal subunit from Haloarcula marismortui, we have identified 116 magnesium ions and 88 monovalent cations bound principally to rRNA. Although the rRNA structures to which these metal ions bind are highly idiosyncratic, a few common principles have emerged from the identities of the specific functional groups that coordinate them. The nonbridging oxygen of a phosphate group is the most common inner shell ligand of Mg++, and Mg++ ions having one or two such inner shell ligands are very common. Nonbridging phosphate oxygens and the heteroatoms of nucleotide bases are common outer shell ligands for Mg++ ions. Monovalent cations usually interact with nucleotide bases and protein groups, although some interactions with nonbridging phosphate oxygens are found. The most common monovalent cation binding site is the major groove side of G-U wobble pairs. Both divalent and monovalent cations stabilize the tertiary structure of 23S rRNA by mediating interactions between its structural domains. Bound metal ions are particularly abundant in the region surrounding the peptidyl transferase center, where stabilizing cationic tails of ribosomal proteins are notably absent. This may point to the importance of metal ions for the stabilization of specific RNA structures in the evolutionary period prior to the appearance of proteins, and hence many of these metal ion binding sites may be conserved across all phylogenetic kingdoms.     

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.     

Altering the intermediate in the equilibrium folding of unmodified yeast tRNAPhe with monovalent and divalent cations

The isothermal equilibrium folding of the unmodified yeast tRNA(Phe) is studied as a function of Na(+), Mg(2+), and urea concentration with hydroxyl radical protection, circular dichroism, and diethyl pyrocarbonate (DEPC) modification. These assays indicate that this tRNA folds in Na(+) alone. Similar to folding in Mg(2+), folding in Na(+) can be described by two transitions, unfolded-to-intermediate-to-native. The I-to-N transition has a Na(+) midpoint of approximately 0.5 M and a Hill constant of approximately 4. Unexpectedly, the urea m-value, the dependence of free energy on urea concentration, for the I-to-N transition is significantly smaller in Na(+) than in Mg(2+), 0.4 versus 1.7 kcal mol(-1) M(-1), indicating that more structure is formed in the Mg(2+)-induced transition. DEPC modification indicates that the I state in Na(+)-induced folding contains all four helices of tRNA and the I-to-N transition primarily corresponds to the formation of the tertiary structure. In contrast, the intermediate in Mg(2+)-induced folding contains only three helices, and the I-to-N transition corresponds to the formation of the acceptor stem plus tertiary structure. The cation dependence of the intermediates arises from the differences in the stability of the acceptor stem and the tertiary structure. The acceptor stem is stable at a lower Na(+) concentration than required for the tertiary structure formation. The relative stability is reversed in Mg(2+) so that the acceptor stem and the tertiary structure form simultaneously in the I-to-N transition. These results demonstrate that formation of the RNA secondary structure can be independent or coupled to the formation of the tertiary structure depending on their relative stability in monovalent and divalent ions.     

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.     

Stabilization of RNA tertiary structure by monovalent cations

The effects of monovalent cations (Li(+), Na(+), K(+), Rb(+), Cs(+), and NH4(+)) on the thermal stability of RNA tertiary structure were investigated by UV melting. We show that with the RNA used here (nucleotides 1051-1108 of Escherichia coli 23 S rRNA with four base substitutions), monovalent cations and Mg(2+) compete in stabilizing the RNA tertiary structure, and that the competition takes place between two boundaries: one where Mg(2+) concentration is zero and the other where it is maximally stabilizing ("saturating"). The pattern of competition is the same for all monovalent cations and depends on the cation's ability to displace Mg(2+) from the RNA, its ability to stabilize tertiary structure in the absence of Mg(2+), and its ability to stabilize tertiary structure at saturating Mg(2+) concentrations. The stabilizing ability of a monovalent cation depends on its unhydrated ionic radius, and at a low monovalent cation concentration and saturating Mg(2+), there is a (calculated) net release of a single monovalent cation/RNA molecule when tertiary structure is denatured. The implications are that under these conditions there is at least one binding site for monovalent cations on the RNA, the site is specifically associated with formation of stable tertiary structure, K(+) is the most effective of the tested cations, and Mg(2+) appears ineffective at this site. At high ionic strength, and in the absence of Mg(2+), stabilization of tertiary structure is still monovalent-cation specific and ionic-radius dependent, but a larger number of cations ( approximately eight) are released upon RNA tertiary structure denaturation, and NH(4)(+) appears to be the most effective cation in stabilizing tertiary structure under these conditions. In the majority of the experiments, methanol was added as a cosolvent to the buffer. Its use allowed the examination of the behavior of monovalent ions under conditions where their effects would otherwise have been too weak to be observed. Methanol stabilizes tertiary but not secondary structure of the RNA. There was no evidence that it either causes qualitative changes in cation-binding properties of the RNA or a change in the pattern of monovalent cation/Mg(2+) competition.     

Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods

RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na(+)]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg(2+) salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs.     

A guide to ions and RNA structure

RNA folding into stable tertiary structures is remarkably sensitive to the concentrations and types of cations present; an understanding of the physical basis of ion-RNA interactions is therefore a prerequisite for a quantitative accounting of RNA stability. This article summarizes the energetic factors that must be considered when ions interact with two different RNA environments. "Diffuse ions" accumulate near the RNA because of the RNA electrostatic field and remain largely hydrated. A "chelated" ion directly contacts a specific location on the RNA surface and is held in place by electrostatic forces. Energetic costs of ion chelation include displacement of some of the waters of hydration by the RNA surface and repulsion of diffuse ions. Methods are discussed for computing both the free energy of the set of diffuse ions associated with an RNA and the binding free energies of individual chelated ions. Such calculations quantitatively account for the effects of Mg(2+) on RNA stability where experimental data are available. An important conclusion is that diffuse ions are a major factor in the stabilization of RNA tertiary structures.     

Ion Condensation onto Ribozyme Is Site Specific and Fold Dependent

The highly charged RNA molecules, with each phosphate carrying a single negative charge, cannot fold into well-defined architectures with tertiary interactions in the absence of ions. For ribozymes, divalent cations are known to be more efficient than monovalent ions in driving them to a compact state, although Mg²⁺ ions are needed for catalytic activities. Therefore, how ions interact with RNA is relevant in understanding RNA folding. It is often thought that most of the ions are territorially and nonspecifically bound to the RNA, as predicted by the counterion condensation theory. Here, we show using simulations of Azoarcus ribozyme, based on an accurate coarse-grained three-site interaction model with explicit divalent and monovalent cations, that ion condensation is highly specific and depends on the nucleotide position. The regions with high coordination between the phosphate groups and the divalent cations are discernible even at very low Mg²⁺ concentrations when the ribozyme does not form tertiary interactions. Surprisingly, these regions also contain the secondary structural elements that nucleate subsequently in the self-assembly of RNA, implying that ion condensation is determined by the architecture of the folded state. These results are in sharp contrast to interactions of ions (monovalent and divalent) with rigid charged rods, in which ion condensation is uniform and position independent. The differences are explained in terms of the dramatic nonmonotonic shape fluctuations in the ribozyme as it folds with increasing Mg²⁺ or Ca²⁺ concentration.     

Existence of efficient divalent metal ion-catalyzed and inefficient divalent metal ion-independent channels in reactions catalyzed by a hammerhead ribozyme

The hammerhead ribozyme is generally accepted as a well characterized metalloenzyme. However, the precise nature of the interactions of the RNA with metal ions remains to be fully defined. Examination of metal ion-catalyzed hammerhead reactions at limited concentrations of metal ions is useful for evaluation of the role of metal ions, as demonstrated in this study. At concentrations of Mn²⁺ ions from 0.3 to 3 mM, addition of the ribozyme to the reaction mixture under single-turnover conditions enhances the reaction with the product reaching a fixed maximum level. Further addition of the ribozyme inhibits the reaction, demonstrating that a certain number of divalent metal ions is required for proper folding and also for catalysis. At extremely high concentrations, monovalent ions, such as Na⁺ ions, can also serve as cofactors in hammerhead ribozyme-catalyzed reactions. However, the catalytic efficiency of monovalent ions is extremely low and, thus, high concentrations are required. Furthermore, addition of monovalent ions to divalent metal ion-catalyzed hammerhead reactions inhibits the divalent metal ion-catalyzed reactions, suggesting that the more desirable divalent metal ion–ribozyme complexes are converted to less desirable monovalent metal ion–ribozyme complexes via removal of divalent metal ions, which serve as a structural support in the ribozyme complex. Even though two channels appear to exist, namely an efficient divalent metal ion-catalyzed channel and an inefficient monovalent metal ion-catalyzed channel, it is clear that, under physiological conditions, hammerhead ribozymes are metalloenzymes that act via the significantly more efficient divalent metal ion-dependent channel. Moreover, the observed kinetic data are consistent with Lilley’s and DeRose’s two-phase folding model that was based on ground state structure analyses.     

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.     

Metal ion binding sites in a group II intron core

Group II introns are catalytic RNA molecules that require divalent metal ions for folding, substrate binding, and chemical catalysis. Metal ion binding sites in the group II core have now been elucidated by monitoring the site-specific RNA hydrolysis patterns of bound ions such as Tb(3+) and Mg(2+). Major sites are localized near active site elements such as domain 5 and its surrounding tertiary interaction partners. Numerous sites are also observed at intron substructures that are involved in binding and potentially activating the splice sites. These results highlight the locations of specific metal ions that are likely to play a role in ribozyme catalysis.     

Multiple folding pathways for the P4-P6 RNA domain

We recently described site-specific pyrene labeling of RNA to monitor Mg(2+)-dependent equilibrium formation of tertiary structure. Here we extend these studies to follow the folding kinetics of the 160-nucleotide P4-P6 domain of the Tetrahymena group I intron RNA, using stopped-flow fluorescence with approximately 1 ms time resolution. Pyrene-labeled P4-P6 was prepared using a new phosphoramidite that allows high-yield automated synthesis of oligoribonucleotides with pyrene incorporated at a specific 2'-amino-2'-deoxyuridine residue. P4-P6 forms its higher-order tertiary structure rapidly, with k(obs) = 15-31 s(-1) (t(1/2) approximately 20-50 ms) at 35 degrees C and [Mg(2+)] approximately 10 mM in Tris-borate (TB) buffer. The folding rate increases strongly with temperature from 4 to 45 degrees C, demonstrating a large activation enthalpy DeltaH(double dagger) approximately 26 kcal/mol; the activation entropy DeltaS(double dagger) is large and positive. In low ionic strength 10 mM sodium cacodylate buffer at 35 degrees C, a slow (t(1/2) approximately 1 s) folding component is also observed. The folding kinetics are both ionic strength- and temperature-dependent; the slow phase vanishes upon increasing [Na(+)] in the cacodylate buffer, and the kinetics switch completely from fast at 30 degrees C to slow at 40 degrees C. Using synchrotron hydroxyl radical footprinting, we confirm that fluorescence monitors the same kinetic events as hydroxyl radical cleavage, and we show that the previously reported slow P4-P6 folding kinetics apply only to low ionic strength conditions. One model to explain the fast and slow folding kinetics postulates that some tertiary interactions are present even without Mg(2+) in the initial state. The fast kinetic phase reflects folding that is facilitated by these interactions, whereas the slow kinetics are observed when these interactions are disrupted at lower ionic strength and higher temperature.