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Metal ion binding to catalytic RNA molecules Victoria J DeRose Cations play critical roles in ribozyme structure and catalysis. Unraveling the contributions of cations as catalytic cofactors is a complex process, due to their role in inducing RNA folding and their potential ability to influence chemical reactions. Recent studies have made progress in separating these roles by directly comparing ion-induced folding with ribozyme activity. In addition, spectroscopic studies have allowed some ribozyme metal sites to be directly observed in solution, providing binding affinities and ligand information. The emerging picture suggests that important cation sites can be classified according to their affinities and properties, and can be located within the ribozyme structure. At moderate ionic strengths, a common theme is emerging for some ribozymes of structural sites that have relatively high metal ion affinities and a second type of metal site with weaker affinity that is responsible for catalysis or structural fine-tuning. In the larger ribozymes, apparent clusters of metal-sensitive positions are observed. Addresses Department of Chemistry, Texas A&M University, College Station, TX 77842-3012, USA e-mail: [email protected]
Current Opinion in Structural Biology 2003, 13:317–324 This review comes from a themed issue on Nucleic acids Edited by Carl C Correll and David MJ Lilley 0959-440X/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-440X(03)00077-0
Abbreviations ENDOR electron nuclear double resonance EPR electron paramagnetic resonance ESEEM electron spin echo envelope modulation HDV hepatitis delta virus PS-rescue phosphorothioate metal-rescue RNase P ribonuclease P SAXS small-angle X-ray scattering
Introduction How does RNA perform chemical reactions? Answers to this question have been sought since the 20-year-old discovery of ‘catalytic’ RNA, or ribozymes [1,2,3]. RNA-catalyzed phosphoryl and aminoacyl transfer reactions are critical to biological function. Four nucleobases encode sequence-specific structure and chemical activity in RNA, and these, in combination with sugar, phosphodiester groups and cofactors such as cations, are the potential players in creating ribozyme active sites. In comparison with proteins, nucleic acid bases provide less www.current-opinion.com
chemical diversity than that available from amino acids, and activity takes place in a background of a more complex folding landscape that is strongly influenced by electrostatics. Ribozymes have been examined using many principles derived from classical enzymology. In parallel, techniques for measuring RNA structure and dynamics have been under development. From the current data, it is evident that the sensitive nature of RNA structure brings new complexities to enzymological studies of ribozymes. Cations, monovalent and divalent, are critical to RNA structure and function [4,5]. The activities of most ribozymes are highly sensitive to ionic conditions and, at moderate ionic strength, most ribozymes require divalent cations such as Mg2þ for activity [3]. As RNA folding depends strongly on electrostatics, cations may be solely required for the formation of the correct RNA structure. On the other hand, metal ions also provide one way of overcoming the dearth of reactive groups on RNA and may be directly involved as cofactors in the chemical reaction. Cations can enable catalysis by activation of the nucleophile (general base catalysis), stabilization of the transition state (charge neutralization) and protonation of the leaving group (general acid catalysis). This review will focus specifically on recent advances concerning our understanding of the influence of cations on ribozyme activities. Two main emphases are presented: first, some recent examples of biochemical methods used to predict specific metal sites in ribozymes; and, second, our current ability to identify and directly examine these metal sites using spectroscopic methods. Initial work on all ribozymes indicated that Mg2þ or other divalent ions were necessary for activity [1,3]. These studies were generally performed in a background of low monovalent ion (Naþ, Kþ) concentration, which alone did not support ribozyme chemistry. ‘Small’ ribozymes, such as the hammerhead, hairpin, Neurospora VS and hepatitis delta virus (HDV) ribozymes, are truncated motifs that catalyze a single site-specific phosphodiester bond cleavage reaction, as shown in Figure 1. Moderate concentrations of monovalent ions ( 1 M) do not promote activity in these ribozymes, but activity rises in micromolar concentrations of Mg2þ or other divalent ions. These results suggested that the ribozymes contain sites that are specific for divalent ions and are critical to catalysis. However, it has now been shown that very high concentrations of monovalent ions, such as 4 M Naþ, Liþ or NH4þ, also support activity in these ribozymes, with rates within an order of magnitude of the maximum observed [6,7]. Thus, the critical cation ‘sites’ that must Current Opinion in Structural Biology 2003, 13:317–324
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Figure 1
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Reactions catalyzed by ribozymes. (a) The reaction catalyzed by the ‘small’ ribozymes (hammerhead, HDV, hairpin and Neurospora VS) and RNase P RNA. (b) The reaction catalyzed by the group I intron ribozyme. The group II intron ribozyme catalyzes a similar reaction with a different extrinsic nucleophile. Ribozyme reactions may require activation of the nucleophile (general base catalysis by B:) and proton donation to the leaving group (general acid, A:H). Metal ions have been proposed to serve either role as metal–aqua or metal–hydroxo species, or to provide electrostatic stabilization of the substrate or transition state. Adapted from [3].
be populated for ribozyme activity can accommodate monovalent ions as well as divalent ions, but at much lower apparent affinities for the former. Working at lower ionic strength, as described below, it is possible to separate the influences of certain types of cation interactions. The nature of these sites, and the question of whether they are indeed discrete cation-binding sites, will be considered in more detail below. In contrast to the small ribozymes, the ‘large’ group I and group II introns, and ribonuclease P (RNaseP) RNA ribozymes are not active in monovalent ions alone, even at very high ionic strengths [1,2,3,4,8,9]. Thus, these ribozymes appear to require Mg2þ or other divalent cations for activity. The group I and group II introns catalyze selfsplicing reactions that initiate with nucleophilic attack by an exogenous hydroxyl group (such as from a sugar 20 - or 30 -OH of an exogenous guanosine bound at the active site) (Figure 1b). RNase P RNA processes 30 termini of tRNA by catalyzing the same reaction as that shown in Figure 1a. It is not immediately apparent why large ribozymes would require multivalent cations for activity when small ribozymes do not, unless their larger size and complexity mandates the use of multivalent ions to direct correct RNA folding. However, as described below, many of Current Opinion in Structural Biology 2003, 13:317–324
the metal sites identified biochemically in the larger ribozymes appear in clusters around their active sites, suggesting more intimate roles in catalysis.
The challenge of assigning specific roles to metals in ribozymes Why would multivalent cations be required for ribozyme catalysis? The two main possibilities are for structure (i.e. folding) and for catalysis. The challenge comes in separating these properties. As noted above, cations provide chemical functionalities that are not available from RNA itself. On the other hand, RNA folding often also has a specific cation requirement. One obvious aid in separating these two influences is to physically locate the site or sites of metal binding on the RNA. Moreover, one would like to correlate population of metal sites to either folding or activity of the ribozyme. The ideal metal-dependent experiments would isolate changes in RNA structure from changes in the chemical reaction. The former is tractable; it is possible to devise experiments that monitor gross changes in RNA structure as a function of added metal ions, using ribozymes inhibited from cleavage by single-atom changes. Beautiful experiments have been performed that map ion-dependent changes in RNA structure using fluorescence resonance energy transfer www.current-opinion.com
Metal ion binding to catalytic RNA molecules DeRose 319
(FRET) [10], footprinting [11,12], small-angle X-ray scattering (SAXS) [9,13] and single-molecule fluorescence techniques [14], as well as thermal denaturation [5,15]. These experiments rarely allow direct location of the metal site(s), but provide instead the overall influence of metals on RNA folding. There are few assays, however, that can achieve the opposite and are guaranteed to report on metal-dependent changes in activity that do not result from an alteration in structure.
sites’ of many ribozymes may be relatively plastic, undergoing conformational changes between ground and pretransition or transition state geometries. Although all enzymes may have this property, the degree of plasticity in ribozymes is not yet fully understood, making the definition of an ‘active site’ and associated ‘catalytic cofactor’ less rigorous. Here, we suggest that a metal ion may be considered directly involved in catalysis if it contributes to stabilizing the transition state and is associated with a group that changes bond order during the reaction. General acid/base catalysis or transition state stabilization through specific cation site binding both fall into this category. Thus, if a metal site identified by PS-rescue is predicted to be far (and stay far) from the active site, the metal site may be considered to be structural. If, however, the rescuable sulfur substitution involves a bond that changes order during the reaction, the metal ion might be directly involved in catalysis.
The phosphorothioate metal-rescue experiment: isolating structure from chemistry? One experiment that has been used extensively to locate metal-ion-binding sites in RNA and that may come close to isolating chemistry from structural changes is the phosphorothioate metal-rescue (PS-rescue) experiment (Figure 2) [16]. This is a special class of single-atom substitution in the general category of nucleotide analog interference mapping (NAIM) and is used the most often to predict RNA metal sites. The PS-rescue experiment tests the hypothesis that a metal ion binds directly to a particular phosphate oxygen and that the metal ion is important to ribozyme function. A nonbridging oxygen can be substituted by sulfur, creating a stereospecific thiophosphate. The substituted site has lost affinity for Mg2þ, but has enhanced affinity for thiophilic ions such as Cd2þ, Zn2þ or Tlþ. Thus, if Mg2þ-dependent activity is lost with a specific oxygen to sulfur substitution, but Cd2þ can rescue activity, a functional metal ion site is predicted.
RNA structure depends on complex networks of hydrogen bonds and other interactions, and even single-atom substitutions might alter local structure, if not cause more extensive changes. The oxygen to sulfur substitution in a phosphorothioate is considered a conservative change. In some cases, however, the longer phosphorus–sulfur bond length, larger ionic radius of sulfur and possible change in charge distribution apparently contribute to a more dramatic change in RNA structure. This has been documented for one Rp substitution in an RNA hairpin [17] and for one out of six sulfur substitutions in a loop region of a GAAA tetraloop [18,19]. However, it has also been shown that, for an RNA duplex, a phosphorothioate substitution does not create a new high-affinity binding site for
As described in more detail below and elsewhere [3], during the course of a standard reaction assay, the ‘active Figure 2
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Metal interactions with ribozyme active sites, as proposed from biochemical experiments following single-atom substitutions. A yellow square denotes sulfur substitutions, and sites in blue squares were tested with amine (b) or deaza (c) substitutions. (a) Metal coordination to the scissile phosphate of the hammerhead ribozyme, as predicted by PS-rescue experiments ([22], reviewed in [3]). The A9 phosphate is also sensitive to PS-rescue and is proposed [22] to provide the metal ion to the scissile phosphate following a conformational change. 31P NMR spectroscopy has provided evidence of metal coordination to A9, but not to the scissile phosphate [20,23]; one possible explanation is a requirement for the metal–OH interaction denoted by (?). A smaller, pH-dependent conformational change has been predicted from X-ray crystallographic studies [21]. (b) Three metal ions predicted for the group I intron [27]. (c) Metal sites predicted from single-atom substitution studies of RNase P RNA [28]; the (?) symbol denotes ambiguity in evidence of the third metal ion. The two Mg2þ ions separated by the double-sided arrow are influenced by each other and may be bridged. www.current-opinion.com
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thiophilic metal ions [20]. Thus, Cd2þ PS-rescue experiments that revive ribozyme activity seem likely to report native metal sites, although the experiment may not describe the exact native metal ligands. Because phosphorothioate substitutions are not completely innocent of structure perturbation, one would like independent confirmation of metal sites that are predicted from PS-rescue experiments. This has been achieved by direct spectroscopic methods in just a few reported cases. The first was for the A9 Rp PS substitution in the hammerhead ribozyme [20]. This metal site, which has also been observed by X-ray crystallography [21], includes interactions with the phosphate 50 to A9 and with the imino N7 of G10.1. The A9 Rp substitution knocks out Mg2þ-dependent activity, which can be rescued by Cd2þ [22]. Using 31P NMR, Maderia et al. [20] were able to observe chemical shifts attributed to highaffinity Cd2þ binding to the A9 Rp phosphorothioate within the context of a hammerhead ribozyme inhibited at the cleavage site. The apparent affinity of Cd2þ for this site was similar to that predicted from activity studies, confirming that metal coordination to the A9 position was actually influencing ribozyme activity. Metal coordination was also observed by Taira and co-workers for a duplex model containing two sheared G A base pairs that mimics the A9/G10.1 coordination site [23,24]. This same 31 P NMR method was used by Butcher and co-workers [25] to confirm metal coordination to an Sp phosphorothioate 50 to U80 in the spliceosome U6 RNA. Metal coordination to the U80 phosphate was predicted to be required for the first step in pre-mRNA splicing, based on PS-interference experiments [26]. Of note, the functional role of this spliceosomal metal site, whether structural or catalytic, is not yet certain. The actual cleavage site of the hammerhead ribozyme provides an interesting exception to the success of 31P NMR methods in directly confirming metal sites predicted from PS-interference studies. There is a clear inhibition of Mg2þ-dependent activity when the pro-R oxygen at the hammerhead ribozyme scissile phosphate is substituted by sulfur and a clean rescue of activity for this Rp substitution in the presence of either Mn2þ or Cd2þ [22]. However, despite this prediction from activity studies, and unlike the case of the hammerhead A9 phosphate, 31P NMR spectroscopy of an Rp phosphorothioate at the hammerhead cleavage site predicts either very weak or no interaction of Cd2þ [20,23]. The reasons for this discrepancy, although not entirely clear, support subtleties that appear from careful analyses of the PS-rescue experiments. Based on apparent binding constants derived from these rescue experiments, it has been proposed that metals do not actually bind to the hammerhead cleavage site in the ground state structure of the ribozyme [22,23]. Rather, from this and other studies [3,21,22], a conformational change to a structure more Current Opinion in Structural Biology 2003, 13:317–324
closely resembling the transition state is predicted to precede the hammerhead chemical step. The cleavage site metal interaction is proposed to take place following this structural change, the magnitude of which remains controversial. If the cleavage site metal ion interaction is not present in the dominantly populated ground state ribozyme structure, it may be difficult to observe using spectroscopic methods, even though it is clearly predicted from the PS-rescue profile. An alternative explanation must be noted, however. In the spectroscopic studies, the ribozyme cleavage site has been inhibited by substitutions of the nucleophilic 20 -OH group with 20 -OMe or 20 -deoxy. These inhibitory substitutions could prevent metal binding, if metal coordination at the scissile phosphate also requires a ligand from the 20 position (shown in Figure 2a). Thus, a different and more conservative 20 substitution would be useful to confirm the negative spectroscopic result concerning ground state metal coordination to the hammerhead scissile phosphate. A constellation of metal ions has been predicted at the active site of the group I intron, based on phosphorothioate, phosphorothiolate and 20 -NH2 substitutions whose inhibition of activity could be rescued with transition metal ions [27] (Figure 2b). Similarly, several metal interactions are predicted at the active site of the RNase P RNA [28], from phosphorothioate and other substitutions (Figure 2c), and also at the group II intron active site from cleavage experiments with Tb3þ [29] and support from NMR spectroscopy [4], as well as single-atom substitutions [30]. None of these interesting predictions have yet been directly observed using spectroscopic methods. In the case of the group I intron 20 -NH2 substitution, however, the apparent affinity of Mn2þ for the site affecting the ribozyme rate was increased 102 over the apparent affinity of Mn2þ for the native 20 -OH ribozyme [27]. Such a change in apparent affinity is expected when an Mn–OH ligand interaction is substituted by Mn–amine coordination, and lends support to the model of a direct metal interaction at that site. Under favorable circumstances, this metal interaction might be confirmed using one of the electron paramagnetic resonance (EPR) methods described below.
Structural analysis of metal sites in RNA using EPR spectroscopy There is a rich history of using spectroscopic methods to study metal sites in proteins. In metalloenzymes, activity is often highly affected by small changes in the ligands of critical metal sites. Spectroscopic methods allow these changes to be directly observed and then correlated to the tuning of enzymatic activity. Just as methods have been adapted to allow ribozymes to be studied by enzymological methods, in favorable cases the metal sites in ribozymes might be amenable to spectroscopic analyses. Such has been the case for the A9/G10.1 metal ion site in the hammerhead ribozyme [31,32] (Figure 3). At moderate www.current-opinion.com
Metal ion binding to catalytic RNA molecules DeRose 321
Figure 3
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Ligands to Mn2þ in the A9/G10.1 site of the hammerhead ribozyme. Metal binding to this site has been established using EPR methods [30,31] and shown to have an affinity of Kd 10 mM in 1 M NaCl. Aqua ligands, a guanine and inner-sphere coordination to phosphate were identified using ENDOR and ESEEM spectroscopic methods. The hammerhead ribozyme enzyme strand is shown in cyan, the substrate strand is in blue and conserved ‘core’ nucleotides are in red.
ionic strength, modifications of the A9 pro-R oxygen and the G10.1 N7 position cause dramatic decline in ribozyme activity, and metal coordination at this site is predicted from X-ray crystallography. To understand the influence of this site, it is important to know the apparent affinity of metals for the A9/G10.1 site under solution conditions at different monovalent ion concentrations and whether these affinities correlate at all with the concentration of metals required to achieve full hammerhead activity. Moreover, the exact ligands to the metal ion under solution conditions are of interest. Some of these goals have been achieved through the use of paramagnetic Mn2þ ions and their examination by EPR spectroscopy. Mn2þ is similar to Mg2þ in ionic radius and enthalpy of hydration, and supports higher hammerhead ribozyme rates in 0.1–1.0 M NaCl. The EPR signal of Mn2þ (S ¼ 5/2) is a distinctive six-line pattern that has two convenient properties: when bound to RNA, the Mn2þ EPR signal changes, allowing Mn2þ binding to be quantified; moreover, Mn2þ bound to RNA has a unique EPR signal that can be further probed for coordination details. Using these methods, a Mn2þ site with high apparent affinity (Kd 10 mM in 1 M NaCl) was www.current-opinion.com
detected in the hammerhead ribozyme (reviewed in [3]). This site should be nearly uniquely populated in a 1:1 ratio of Mn2þ to RNA at concentrations >100 mM and indeed, under these conditions, the Mn2þ EPR signal shows subtle line-shape changes indicating that the metal is bound to the ribozyme [31]. Given an EPR signature for bound Mn2þ, this hammerhead ribozyme metal site could be further examined through hyperfine interactions between the nuclei of the RNA ligands and the Mn2þ electron spin. In RNA, natural abundance 31P (phosphodiesters), 1H (exchangeable, on aqua ligands, and nonexchangeable, on sugars and bases) and 14N (nucleobases) are all candidates for ligand identification based on nuclear spin [31,32,33]. Isotopic labeling with 13C or 2H can also be used. Hyperfine interactions can be observed using ENDOR (electron nuclear double resonance) and ESEEM (electron spin echo envelope modulation) experiments, both of which rely on observing the influence of nearby nuclear spins on the Mn2þ EPR signal. For the tight Mn2þ site in the hammerhead ribozyme, ENDOR allowed the identification of directly coordinated phosphate and aqua ligands [31] (Figure 3). The N7 of a guanine base was Current Opinion in Structural Biology 2003, 13:317–324
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detected via ESEEM spectroscopy in combination with a 15 N-guanine-labeled hammerhead enzyme strand [32]. Taken together, these experiments strongly suggested that the unique Mn2þ site in the hammerhead ribozyme is indeed the A9/G10.1 site; this result has recently been confirmed with site-specific 15N labeling (M Vogt, S Lahiri, CG Hoogstraten, RD Britt, VJ DeRose, unpublished data). Thus, a single specific metal site with high affinity can be populated in this 47-nucleotide RNA, even in a background of 1 M NaCl. As noted above, this same site was also probed using 31P NMR with a phosphorothioate at the A9 position. These techniques provide a signal from a functional metal site whose population modulates ribozyme activity.
Relating metal sites to ribozyme function The advantage of a clear signal from a metal site is that it allows correlation of the population of that particular site with the appearance of ribozyme activity. In the case of the hammerhead ribozyme, knowing the apparent affinity of the A9/G10.1 site allows us to conclude that at least two types of metal sites influence hammerhead activity. This conclusion is reached because, at 1 M NaCl, the A9/G10.1 site (Kd 10 mM) is populated at Mn2þ concentrations much lower than the >1 mM Mn2þ required for full activity. Thus, even though the PS-rescue experiment shows that hammerhead activity is sensitive to the high-affinity metal site, at least one other weaker site(s) is also required. The picture of high- and low-affinity cation interactions being required for ribozyme function has been echoed by other recent studies. Hampel and Burke [12] have very recently compared the cation requirements of hammerhead ribozyme folding and activity. Folding, as measured by solvent protection in a hydroxyl radical footprinting experiment, takes place at cation concentrations signif-
icantly lower than those required for catalysis, with Mg2þ or Liþ as the supporting cation. Interestingly, a similar situation has recently been described for the HDV ribozyme. Using thermal denaturation as a measure of ribozyme folding, Bevilacqua and co-workers [15] propose a structural Mg2þ site(s) (Hill coefficient 1) with an apparent affinity much higher than that of a catalytic Mg2þ interaction. From their work on the folding of RNase P RNA, Fang et al. [9] predict that a final rearrangement from a relatively stable folding intermediate to the active form of the ribozyme involves small structural changes around a specific metal ion site. These studies point to an overall picture of ribozymes in which population of highaffinity cation sites leads to relatively large structural rearrangements. The final fine-tuning of an active site, however, may require population of cation site(s) with relatively weak affinity. Such a situation is depicted schematically in Figure 4. Whether a final ‘fine-tuning’ of ribozyme activities by cations with relatively weak affinities makes the cations ‘catalytic’ cofactors may be a question of nomenclature. At the beginning of this review, ‘catalytic’ metal ions were defined as those that contact groups that change bond order during the chemical reaction. It is certainly the case that thiophilic metal ions that rescue deleterious effects of sulfur substitutions at ribozyme active sites fall within this definition of catalytic cofactors. However, even slightly noninnocent single-atom substitutions such as thiophosphates may be able to change the rate-limiting step in ribozyme kinetics. Hard-core enzymologists may prefer the gold standard of isotope effects, whereby the substitution has no structural effect. Direct spectroscopic probes, as well as a better understanding of the effects on RNA structure of substitutions used in ribozyme studies, will aid future investigations of ribozyme activity.
Figure 4
Ma (tight) structural
Mb (weak) structural or catalytic Current Opinion in Structural Biology
Model of classes of metal sites that influence ribozyme activity. As described in the text, in the hammerhead and HDV ribozymes, evidence has been provided of high-affinity structural metal ion sites (Ma) and weaker-affinity metal sites that influence catalysis (Mb). The weaker-affinity sites may directly coordinate and influence moieties involved in the chemical reaction, or may foster small structural changes near the active site that are critical to ribozyme chemistry. The arrow on the right indicates the cleavage site. Current Opinion in Structural Biology 2003, 13:317–324
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Metal ion binding to catalytic RNA molecules DeRose 323
Conclusions In this review, some current studies on the effects of cations on ribozyme activity have been presented. It has been pointed out that separating cation influences on RNA structure from direct involvement in the chemical step of ribozyme reactions is a significant challenge. For large ribozymes, RNA positions whose substitutions affect metal-dependent activity appear in clusters around the ribozyme active site. This suggests that metals aid in the reactions of large ribozymes by directly contacting reactive groups, although the exact details of such contacts remain to be determined for the native RNAs. In the hammerhead and HDV ribozymes, metal-induced folding requires population of cation sites with higher affinities than those required for ribozyme chemistry, leading to a general picture of at least two classes of cation sites. In the hammerhead ribozyme, at least one metal site can be discretely populated, examined in situ and have a strong influence on activity, suggesting a fine-tuning of ribozyme function by a cation cofactor. It is evident that RNA has evolved more than one method of harnessing electrostatics to influence its function. Finally, it is important to note that, for at least two of the small ribozymes, although cations certainly influence function, the majority of the catalytic power is probably derived from nucleobases [34]. The possible interplay between cation sites and base pKas has been noted [15,25].
Acknowledgements The author wishes to thank the Section editors, Carl C Correll and David MJ Lilley, for helpful comments. Support from the NIH (GM58096), NSF (CHE0111696) and THECB is gratefully acknowledged.
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. Doudna J, Cech T: The chemical repertoire of natural ribozymes. Nature 2002, 418:222-228. A review of the first 20 years of ribozyme investigations. 2. Fedor MJ, Westhof E: Ribozymes: the first 20 years. Mol Cell 2002, 10:703-704. A brief but important account of news from a meeting held in celebration of the 20-year anniversary of ribozymes. 3.
DeRose VJ: Two decades of RNA catalysis. Chem Biol 2002, 9:961-969.
4. Pyle AM: Metal ions in the structure and function of RNA. J Biol Inorg Chem 2002, 7:679-690. This article reviews structural aspects of known metal sites in RNA and their influences on function. It concludes with a review of work that identified a cluster of metal interactions with the D5 helix of the group II intron.
reactions stimulated by monovalent and divalent cations. RNA 2001, 7:537-545. This article, one of a few that characterize the activity of the small ribozymes in high concentrations of monovalent cations, describes a careful comparison of the effect of many different single-atom substitutions on hammerhead ribozyme activity under different ionic conditions. The authors conclude that the majority of substitutions have the same effect for both monovalent and divalent cations, with some interesting exceptions of phosphorothioates at the A9 and cleavage sites, and that therefore the overall mechanism is very similar under very different cation backgrounds. 7.
Nakano S, Proctor DJ, Bevilacqua PC: Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism. Biochemistry 2001, 40:12022-12038.
8.
Sigel R, Vaidya A, Pyle A: Metal ion binding sites in a group II intron core. Nat Struct Biol 2000, 7:1111-1116.
9.
Fang X-W, Thiyagarajan P, Sosnick TR, Pan T: The rate-limiting step in the folding of a large ribozyme without kinetic traps. Proc Natl Acad Sci USA 2002, 99:8518-8523. In this work, the folding of the RNase P RNA is investigated kinetically and by SAXS techniques. Evidence is provided of a rate-limiting step in the folding pathway that depends on a relatively small, but energetically significant, rearrangement of a metal ion already bound in a previous step. 10. Hammann C, Lilley DMJ: Folding and activity of the hammerhead ribozyme. Chembiochem 2002, 3:690-700. 11. Rangan P, Masquida B, Westhof E, Woodson SA: Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme. Proc Natl Acad Sci USA 2003, 100:1574-1579. A recent and characteristically elegant example of time-resolved hydroxyl radical footprinting techniques applied to metal-dependent RNA folding. 12. Hampel KJ, Burke JM: Solvent protection of the hammerhead ribozyme in the ground state: evidence for a cation-assisted conformational change leading to catalysis. Biochemistry 2003, 42:4421-4429. This paper describes a direct comparison of metal-dependent RNA folding, probed by hydroxyl radical footprinting experiments, with the Mg2þ or Liþ requirements for hammerhead ribozyme catalysis. In both cases, the concentration of metal required for RNA folding is significantly lower than that required for catalysis. 13. Russell R, Millettt IS, Tate MW, Kwok LW, Nakatani B, Gruner SM, Mochrie SGJ, Pande V, Doniach S, Herschlag D, Pollack L: Rapid compaction during RNA folding. Proc Natl Acad Sci USA 2002, 99:4266-4271. SAXS provides a global picture of ion-induced folding in the Tetrahymena group I intron ribozyme. 14. Zhuang X, Rief M: Single-molecule folding. Curr Opin Struct Biol 2003, 13:88-97. A recent review that includes the pioneering single-molecule fluorescence studies of folding in the hairpin ribozyme. 15. Nakano S, Cerrone A, Bevilacqua P: Mechanistic characterization of the HDV genomic ribozyme: classifying the catalytic and structural metal ion sites within a multichannel reaction mechanism. Biochemistry 2003, 42:2982-2994. In another careful examination of ribozyme activity as a function of cation background, Nakano et al. established three channels for HDV ribozyme activity that differ slightly in cation influence. Here, by populating different channels, the characteristics of a structural and a ‘catalytic’ metal are established through both activity and thermal denaturation studies. 16. Eckstein F: Developments in RNA chemistry, a personal view. Biochimie 2002, 84:841-848.
5. Misra VK, Draper DE: The linkage between magnesium binding and RNA folding. J Mol Biol 2002, 317:507-521. In this paper and references therein, Misra and Draper lay out a thermodynamic framework for metal interactions with RNA in terms of several different contributions, the balance of which defines apparent affinities for both specific and nonspecific cation sites. The results from Poisson– Boltzmann calculations are in good agreement with experimental parameters.
17. Smith JS, Nikonowicz EP: Phosphorothioate substitution can substantially alter RNA conformation. Biochemistry 2000, 39:5642-5652.
6.
19. Horton TE, Maderia M, DeRose VJ: Impact of phosphorothioate substitutions on the thermodynamic stability of an RNA GAAA
O’Rear JL, Wang SL, Feig AL, Beigelman L, Uhlenbeck OC, Herschlag D: Comparison of the hammerhead cleavage
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18. Maderia M, Horton TE, DeRose VJ: Metal interactions with a GAAA RNA tetraloop characterized by P-31 NMR and phosphorothioate substitutions. Biochemistry 2000, 39:8190-8200.
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tetraloop: an unexpected stabilization. Biochemistry 2000, 39:8201-8207. 20. Maderia M, Hunsicker LM, DeRose VJ: Metal-phosphate interactions in the hammerhead ribozyme observed by P-31 NMR and phosphorothioate substitutions. Biochemistry 2000, 39:12113-12120. 31 P NMR spectroscopy was used to directly probe Cd2þ interactions with phosphorothioate-substituted sites in the hammerhead ribozyme. As expected from metal-rescue experiments, high-affinity coordination to the hammerhead A9 position is observed. Unexpectedly, weak to little interaction with the cleavage site phosphorothioate is detected. 21. Murray J, Dunham C, Scott W: A pH-dependent conformational change, rather than the chemical step, appears to be ratelimiting in the hammerhead ribozyme cleavage reaction. J Mol Biol 2002, 315:121-130. An X-ray crystallographic study of hammerhead ribozyme structures trapped by methods designed to capture kinetic intermediates. The resulting model suggests a small but critical pH-dependent conformational change that precedes phosphate cleavage. 22. Wang S, Karbstein K, Peracchi A, Beigelman L, Herschlag D: Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate. Biochemistry 1999, 73:14363-14378. 23. Suzumura K, Yoshinari K, Tanaka Y, Takagi Y, Kasai Y, Warashina M, Kuwabara T, Orita M, Taira K: A reappraisal, based on 31P NMR, of the direct coordination of a metal ion with the phosphoryl oxygen at the cleavage site of a hammerhead ribozyme. J Am Chem Soc 2002, 124:8230-8236. 24. Tanaka Y, Kojima C, Morita E, Kasai Y, Yamasaki K, Ono A, Kainosho M, Taira K: Identification of the metal ion binding site on an RNA motif from hammerhead ribozymes using 15 N NMR spectroscopy. J Am Chem Soc 2002, 124:4595-4601.
27. Shan S, Kravchuk AV, Piccirilli JA, Herschlag D: Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction. Biochemistry 2001, 40:5161-5171. Kinetic analyses of several different single-atom substitutions around the active site of the group I intron provide evidence of the model shown in Figure 2. Several metal interactions are predicted. 28. Christian EL, Kaye NM, Harris ME: Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA. EMBO J 2002, 21:2253-2262. In this paper, several different phosphorothioate and base substitutions are explored on or near the P4 helix of RNase P RNA, which is thought to create the active site in cleaving the 30 end of tRNA. Evidence of the set of metal ions shown in Figure 2 is presented. 29. Sigel RKO, Vaidya A, Pyle AM: Metal ion binding sites in a group II intron core. Nat Struct Biol 2000, 7:1111-1116. 30. Gordon PM, Sontheimer EJ, Piccirilli JA: Kinetic characterization of the second step of group II intron splicing: role of metal ions and the cleavage site 20 -OH in catalysis. Biochemistry 2000, 39:12939-12952. 31. Morrissey SR, Horton TE, DeRose VJ: Mn2þ sites in the hammerhead ribozyme investigated by EPR and continuouswave Q-band ENDOR spectroscopies. J Am Chem Soc 2000, 122:3473-3481. This is the first description of a metal site using ‘advanced’ EPR techniques to identify RNA ligands. EPR and ENDOR spectroscopy show that a high-affinity Mn2þ site in the hammerhead ribozyme can be populated, exhibits a perturbed EPR signal and can be probed to extract evidence of phosphate and aqua ligands, as well as a nucleobase. 32. Morrissey SR, Horton TE, Grant CV, Hoogstraten CG, Britt RD, DeRose VJ: Mn2þ-nitrogen interactions in RNA probed by electron spin-echo envelope modulation spectroscopy: application to the hammerhead ribozyme. J Am Chem Soc 1999, 121:9215-9218.
25. Huppler A, Nikstad LJ, Allmann AM, Brow DA, Butcher SE: Metal binding and base ionization in the U6 RNA intramolecular stem-loop structure. Nat Struct Biol 2002, 9:431-435.
33. Hoogstraten CG, Grant CV, Horton TE, DeRose VJ, Britt RD: Structural analysis of metal ion ligation to nucleotides and nucleic acids using pulsed EPR spectroscopy. J Am Chem Soc 2002, 124:834-842.
26. Yean SL, Wuenschell G, Termini J, Lin RJ: Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome. Nature 2000, 408:881-884.
34. Bevilacqua PC: Mechanistic considerations for general acidbase catalysis by RNA: revisiting the mechanism of the hairpin ribozyme. Biochemistry 2003, 42:2259-2265.
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Metal ion binding to catalytic RNA molecules Victoria J DeRose Cations play critical roles in ribozyme structure and catalysis. Unraveling the contributions of cations as catalytic cofactors is a complex process, due to their role in inducing RNA folding and their potential ability to influence chemical reactions. Recent studies have made progress in separating these roles by directly comparing ion-induced folding with ribozyme activity. In addition, spectroscopic studies have allowed some ribozyme metal sites to be directly observed in solution, providing binding affinities and ligand information. The emerging picture suggests that important cation sites can be classified according to their affinities and properties, and can be located within the ribozyme structure. At moderate ionic strengths, a common theme is emerging for some ribozymes of structural sites that have relatively high metal ion affinities and a second type of metal site with weaker affinity that is responsible for catalysis or structural fine-tuning. In the larger ribozymes, apparent clusters of metal-sensitive positions are observed. Addresses Department of Chemistry, Texas A&M University, College Station, TX 77842-3012, USA e-mail: [email protected]
Current Opinion in Structural Biology 2003, 13:317–324 This review comes from a themed issue on Nucleic acids Edited by Carl C Correll and David MJ Lilley 0959-440X/03/$ – see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-440X(03)00077-0
Abbreviations ENDOR electron nuclear double resonance EPR electron paramagnetic resonance ESEEM electron spin echo envelope modulation HDV hepatitis delta virus PS-rescue phosphorothioate metal-rescue RNase P ribonuclease P SAXS small-angle X-ray scattering
Introduction How does RNA perform chemical reactions? Answers to this question have been sought since the 20-year-old discovery of ‘catalytic’ RNA, or ribozymes [1,2,3]. RNA-catalyzed phosphoryl and aminoacyl transfer reactions are critical to biological function. Four nucleobases encode sequence-specific structure and chemical activity in RNA, and these, in combination with sugar, phosphodiester groups and cofactors such as cations, are the potential players in creating ribozyme active sites. In comparison with proteins, nucleic acid bases provide less www.current-opinion.com
chemical diversity than that available from amino acids, and activity takes place in a background of a more complex folding landscape that is strongly influenced by electrostatics. Ribozymes have been examined using many principles derived from classical enzymology. In parallel, techniques for measuring RNA structure and dynamics have been under development. From the current data, it is evident that the sensitive nature of RNA structure brings new complexities to enzymological studies of ribozymes. Cations, monovalent and divalent, are critical to RNA structure and function [4,5]. The activities of most ribozymes are highly sensitive to ionic conditions and, at moderate ionic strength, most ribozymes require divalent cations such as Mg2þ for activity [3]. As RNA folding depends strongly on electrostatics, cations may be solely required for the formation of the correct RNA structure. On the other hand, metal ions also provide one way of overcoming the dearth of reactive groups on RNA and may be directly involved as cofactors in the chemical reaction. Cations can enable catalysis by activation of the nucleophile (general base catalysis), stabilization of the transition state (charge neutralization) and protonation of the leaving group (general acid catalysis). This review will focus specifically on recent advances concerning our understanding of the influence of cations on ribozyme activities. Two main emphases are presented: first, some recent examples of biochemical methods used to predict specific metal sites in ribozymes; and, second, our current ability to identify and directly examine these metal sites using spectroscopic methods. Initial work on all ribozymes indicated that Mg2þ or other divalent ions were necessary for activity [1,3]. These studies were generally performed in a background of low monovalent ion (Naþ, Kþ) concentration, which alone did not support ribozyme chemistry. ‘Small’ ribozymes, such as the hammerhead, hairpin, Neurospora VS and hepatitis delta virus (HDV) ribozymes, are truncated motifs that catalyze a single site-specific phosphodiester bond cleavage reaction, as shown in Figure 1. Moderate concentrations of monovalent ions ( 1 M) do not promote activity in these ribozymes, but activity rises in micromolar concentrations of Mg2þ or other divalent ions. These results suggested that the ribozymes contain sites that are specific for divalent ions and are critical to catalysis. However, it has now been shown that very high concentrations of monovalent ions, such as 4 M Naþ, Liþ or NH4þ, also support activity in these ribozymes, with rates within an order of magnitude of the maximum observed [6,7]. Thus, the critical cation ‘sites’ that must Current Opinion in Structural Biology 2003, 13:317–324
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Figure 1
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Reactions catalyzed by ribozymes. (a) The reaction catalyzed by the ‘small’ ribozymes (hammerhead, HDV, hairpin and Neurospora VS) and RNase P RNA. (b) The reaction catalyzed by the group I intron ribozyme. The group II intron ribozyme catalyzes a similar reaction with a different extrinsic nucleophile. Ribozyme reactions may require activation of the nucleophile (general base catalysis by B:) and proton donation to the leaving group (general acid, A:H). Metal ions have been proposed to serve either role as metal–aqua or metal–hydroxo species, or to provide electrostatic stabilization of the substrate or transition state. Adapted from [3].
be populated for ribozyme activity can accommodate monovalent ions as well as divalent ions, but at much lower apparent affinities for the former. Working at lower ionic strength, as described below, it is possible to separate the influences of certain types of cation interactions. The nature of these sites, and the question of whether they are indeed discrete cation-binding sites, will be considered in more detail below. In contrast to the small ribozymes, the ‘large’ group I and group II introns, and ribonuclease P (RNaseP) RNA ribozymes are not active in monovalent ions alone, even at very high ionic strengths [1,2,3,4,8,9]. Thus, these ribozymes appear to require Mg2þ or other divalent cations for activity. The group I and group II introns catalyze selfsplicing reactions that initiate with nucleophilic attack by an exogenous hydroxyl group (such as from a sugar 20 - or 30 -OH of an exogenous guanosine bound at the active site) (Figure 1b). RNase P RNA processes 30 termini of tRNA by catalyzing the same reaction as that shown in Figure 1a. It is not immediately apparent why large ribozymes would require multivalent cations for activity when small ribozymes do not, unless their larger size and complexity mandates the use of multivalent ions to direct correct RNA folding. However, as described below, many of Current Opinion in Structural Biology 2003, 13:317–324
the metal sites identified biochemically in the larger ribozymes appear in clusters around their active sites, suggesting more intimate roles in catalysis.
The challenge of assigning specific roles to metals in ribozymes Why would multivalent cations be required for ribozyme catalysis? The two main possibilities are for structure (i.e. folding) and for catalysis. The challenge comes in separating these properties. As noted above, cations provide chemical functionalities that are not available from RNA itself. On the other hand, RNA folding often also has a specific cation requirement. One obvious aid in separating these two influences is to physically locate the site or sites of metal binding on the RNA. Moreover, one would like to correlate population of metal sites to either folding or activity of the ribozyme. The ideal metal-dependent experiments would isolate changes in RNA structure from changes in the chemical reaction. The former is tractable; it is possible to devise experiments that monitor gross changes in RNA structure as a function of added metal ions, using ribozymes inhibited from cleavage by single-atom changes. Beautiful experiments have been performed that map ion-dependent changes in RNA structure using fluorescence resonance energy transfer www.current-opinion.com
Metal ion binding to catalytic RNA molecules DeRose 319
(FRET) [10], footprinting [11,12], small-angle X-ray scattering (SAXS) [9,13] and single-molecule fluorescence techniques [14], as well as thermal denaturation [5,15]. These experiments rarely allow direct location of the metal site(s), but provide instead the overall influence of metals on RNA folding. There are few assays, however, that can achieve the opposite and are guaranteed to report on metal-dependent changes in activity that do not result from an alteration in structure.
sites’ of many ribozymes may be relatively plastic, undergoing conformational changes between ground and pretransition or transition state geometries. Although all enzymes may have this property, the degree of plasticity in ribozymes is not yet fully understood, making the definition of an ‘active site’ and associated ‘catalytic cofactor’ less rigorous. Here, we suggest that a metal ion may be considered directly involved in catalysis if it contributes to stabilizing the transition state and is associated with a group that changes bond order during the reaction. General acid/base catalysis or transition state stabilization through specific cation site binding both fall into this category. Thus, if a metal site identified by PS-rescue is predicted to be far (and stay far) from the active site, the metal site may be considered to be structural. If, however, the rescuable sulfur substitution involves a bond that changes order during the reaction, the metal ion might be directly involved in catalysis.
The phosphorothioate metal-rescue experiment: isolating structure from chemistry? One experiment that has been used extensively to locate metal-ion-binding sites in RNA and that may come close to isolating chemistry from structural changes is the phosphorothioate metal-rescue (PS-rescue) experiment (Figure 2) [16]. This is a special class of single-atom substitution in the general category of nucleotide analog interference mapping (NAIM) and is used the most often to predict RNA metal sites. The PS-rescue experiment tests the hypothesis that a metal ion binds directly to a particular phosphate oxygen and that the metal ion is important to ribozyme function. A nonbridging oxygen can be substituted by sulfur, creating a stereospecific thiophosphate. The substituted site has lost affinity for Mg2þ, but has enhanced affinity for thiophilic ions such as Cd2þ, Zn2þ or Tlþ. Thus, if Mg2þ-dependent activity is lost with a specific oxygen to sulfur substitution, but Cd2þ can rescue activity, a functional metal ion site is predicted.
RNA structure depends on complex networks of hydrogen bonds and other interactions, and even single-atom substitutions might alter local structure, if not cause more extensive changes. The oxygen to sulfur substitution in a phosphorothioate is considered a conservative change. In some cases, however, the longer phosphorus–sulfur bond length, larger ionic radius of sulfur and possible change in charge distribution apparently contribute to a more dramatic change in RNA structure. This has been documented for one Rp substitution in an RNA hairpin [17] and for one out of six sulfur substitutions in a loop region of a GAAA tetraloop [18,19]. However, it has also been shown that, for an RNA duplex, a phosphorothioate substitution does not create a new high-affinity binding site for
As described in more detail below and elsewhere [3], during the course of a standard reaction assay, the ‘active Figure 2
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Metal interactions with ribozyme active sites, as proposed from biochemical experiments following single-atom substitutions. A yellow square denotes sulfur substitutions, and sites in blue squares were tested with amine (b) or deaza (c) substitutions. (a) Metal coordination to the scissile phosphate of the hammerhead ribozyme, as predicted by PS-rescue experiments ([22], reviewed in [3]). The A9 phosphate is also sensitive to PS-rescue and is proposed [22] to provide the metal ion to the scissile phosphate following a conformational change. 31P NMR spectroscopy has provided evidence of metal coordination to A9, but not to the scissile phosphate [20,23]; one possible explanation is a requirement for the metal–OH interaction denoted by (?). A smaller, pH-dependent conformational change has been predicted from X-ray crystallographic studies [21]. (b) Three metal ions predicted for the group I intron [27]. (c) Metal sites predicted from single-atom substitution studies of RNase P RNA [28]; the (?) symbol denotes ambiguity in evidence of the third metal ion. The two Mg2þ ions separated by the double-sided arrow are influenced by each other and may be bridged. www.current-opinion.com
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thiophilic metal ions [20]. Thus, Cd2þ PS-rescue experiments that revive ribozyme activity seem likely to report native metal sites, although the experiment may not describe the exact native metal ligands. Because phosphorothioate substitutions are not completely innocent of structure perturbation, one would like independent confirmation of metal sites that are predicted from PS-rescue experiments. This has been achieved by direct spectroscopic methods in just a few reported cases. The first was for the A9 Rp PS substitution in the hammerhead ribozyme [20]. This metal site, which has also been observed by X-ray crystallography [21], includes interactions with the phosphate 50 to A9 and with the imino N7 of G10.1. The A9 Rp substitution knocks out Mg2þ-dependent activity, which can be rescued by Cd2þ [22]. Using 31P NMR, Maderia et al. [20] were able to observe chemical shifts attributed to highaffinity Cd2þ binding to the A9 Rp phosphorothioate within the context of a hammerhead ribozyme inhibited at the cleavage site. The apparent affinity of Cd2þ for this site was similar to that predicted from activity studies, confirming that metal coordination to the A9 position was actually influencing ribozyme activity. Metal coordination was also observed by Taira and co-workers for a duplex model containing two sheared G A base pairs that mimics the A9/G10.1 coordination site [23,24]. This same 31 P NMR method was used by Butcher and co-workers [25] to confirm metal coordination to an Sp phosphorothioate 50 to U80 in the spliceosome U6 RNA. Metal coordination to the U80 phosphate was predicted to be required for the first step in pre-mRNA splicing, based on PS-interference experiments [26]. Of note, the functional role of this spliceosomal metal site, whether structural or catalytic, is not yet certain. The actual cleavage site of the hammerhead ribozyme provides an interesting exception to the success of 31P NMR methods in directly confirming metal sites predicted from PS-interference studies. There is a clear inhibition of Mg2þ-dependent activity when the pro-R oxygen at the hammerhead ribozyme scissile phosphate is substituted by sulfur and a clean rescue of activity for this Rp substitution in the presence of either Mn2þ or Cd2þ [22]. However, despite this prediction from activity studies, and unlike the case of the hammerhead A9 phosphate, 31P NMR spectroscopy of an Rp phosphorothioate at the hammerhead cleavage site predicts either very weak or no interaction of Cd2þ [20,23]. The reasons for this discrepancy, although not entirely clear, support subtleties that appear from careful analyses of the PS-rescue experiments. Based on apparent binding constants derived from these rescue experiments, it has been proposed that metals do not actually bind to the hammerhead cleavage site in the ground state structure of the ribozyme [22,23]. Rather, from this and other studies [3,21,22], a conformational change to a structure more Current Opinion in Structural Biology 2003, 13:317–324
closely resembling the transition state is predicted to precede the hammerhead chemical step. The cleavage site metal interaction is proposed to take place following this structural change, the magnitude of which remains controversial. If the cleavage site metal ion interaction is not present in the dominantly populated ground state ribozyme structure, it may be difficult to observe using spectroscopic methods, even though it is clearly predicted from the PS-rescue profile. An alternative explanation must be noted, however. In the spectroscopic studies, the ribozyme cleavage site has been inhibited by substitutions of the nucleophilic 20 -OH group with 20 -OMe or 20 -deoxy. These inhibitory substitutions could prevent metal binding, if metal coordination at the scissile phosphate also requires a ligand from the 20 position (shown in Figure 2a). Thus, a different and more conservative 20 substitution would be useful to confirm the negative spectroscopic result concerning ground state metal coordination to the hammerhead scissile phosphate. A constellation of metal ions has been predicted at the active site of the group I intron, based on phosphorothioate, phosphorothiolate and 20 -NH2 substitutions whose inhibition of activity could be rescued with transition metal ions [27] (Figure 2b). Similarly, several metal interactions are predicted at the active site of the RNase P RNA [28], from phosphorothioate and other substitutions (Figure 2c), and also at the group II intron active site from cleavage experiments with Tb3þ [29] and support from NMR spectroscopy [4], as well as single-atom substitutions [30]. None of these interesting predictions have yet been directly observed using spectroscopic methods. In the case of the group I intron 20 -NH2 substitution, however, the apparent affinity of Mn2þ for the site affecting the ribozyme rate was increased 102 over the apparent affinity of Mn2þ for the native 20 -OH ribozyme [27]. Such a change in apparent affinity is expected when an Mn–OH ligand interaction is substituted by Mn–amine coordination, and lends support to the model of a direct metal interaction at that site. Under favorable circumstances, this metal interaction might be confirmed using one of the electron paramagnetic resonance (EPR) methods described below.
Structural analysis of metal sites in RNA using EPR spectroscopy There is a rich history of using spectroscopic methods to study metal sites in proteins. In metalloenzymes, activity is often highly affected by small changes in the ligands of critical metal sites. Spectroscopic methods allow these changes to be directly observed and then correlated to the tuning of enzymatic activity. Just as methods have been adapted to allow ribozymes to be studied by enzymological methods, in favorable cases the metal sites in ribozymes might be amenable to spectroscopic analyses. Such has been the case for the A9/G10.1 metal ion site in the hammerhead ribozyme [31,32] (Figure 3). At moderate www.current-opinion.com
Metal ion binding to catalytic RNA molecules DeRose 321
Figure 3
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Ligands to Mn2þ in the A9/G10.1 site of the hammerhead ribozyme. Metal binding to this site has been established using EPR methods [30,31] and shown to have an affinity of Kd 10 mM in 1 M NaCl. Aqua ligands, a guanine and inner-sphere coordination to phosphate were identified using ENDOR and ESEEM spectroscopic methods. The hammerhead ribozyme enzyme strand is shown in cyan, the substrate strand is in blue and conserved ‘core’ nucleotides are in red.
ionic strength, modifications of the A9 pro-R oxygen and the G10.1 N7 position cause dramatic decline in ribozyme activity, and metal coordination at this site is predicted from X-ray crystallography. To understand the influence of this site, it is important to know the apparent affinity of metals for the A9/G10.1 site under solution conditions at different monovalent ion concentrations and whether these affinities correlate at all with the concentration of metals required to achieve full hammerhead activity. Moreover, the exact ligands to the metal ion under solution conditions are of interest. Some of these goals have been achieved through the use of paramagnetic Mn2þ ions and their examination by EPR spectroscopy. Mn2þ is similar to Mg2þ in ionic radius and enthalpy of hydration, and supports higher hammerhead ribozyme rates in 0.1–1.0 M NaCl. The EPR signal of Mn2þ (S ¼ 5/2) is a distinctive six-line pattern that has two convenient properties: when bound to RNA, the Mn2þ EPR signal changes, allowing Mn2þ binding to be quantified; moreover, Mn2þ bound to RNA has a unique EPR signal that can be further probed for coordination details. Using these methods, a Mn2þ site with high apparent affinity (Kd 10 mM in 1 M NaCl) was www.current-opinion.com
detected in the hammerhead ribozyme (reviewed in [3]). This site should be nearly uniquely populated in a 1:1 ratio of Mn2þ to RNA at concentrations >100 mM and indeed, under these conditions, the Mn2þ EPR signal shows subtle line-shape changes indicating that the metal is bound to the ribozyme [31]. Given an EPR signature for bound Mn2þ, this hammerhead ribozyme metal site could be further examined through hyperfine interactions between the nuclei of the RNA ligands and the Mn2þ electron spin. In RNA, natural abundance 31P (phosphodiesters), 1H (exchangeable, on aqua ligands, and nonexchangeable, on sugars and bases) and 14N (nucleobases) are all candidates for ligand identification based on nuclear spin [31,32,33]. Isotopic labeling with 13C or 2H can also be used. Hyperfine interactions can be observed using ENDOR (electron nuclear double resonance) and ESEEM (electron spin echo envelope modulation) experiments, both of which rely on observing the influence of nearby nuclear spins on the Mn2þ EPR signal. For the tight Mn2þ site in the hammerhead ribozyme, ENDOR allowed the identification of directly coordinated phosphate and aqua ligands [31] (Figure 3). The N7 of a guanine base was Current Opinion in Structural Biology 2003, 13:317–324
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detected via ESEEM spectroscopy in combination with a 15 N-guanine-labeled hammerhead enzyme strand [32]. Taken together, these experiments strongly suggested that the unique Mn2þ site in the hammerhead ribozyme is indeed the A9/G10.1 site; this result has recently been confirmed with site-specific 15N labeling (M Vogt, S Lahiri, CG Hoogstraten, RD Britt, VJ DeRose, unpublished data). Thus, a single specific metal site with high affinity can be populated in this 47-nucleotide RNA, even in a background of 1 M NaCl. As noted above, this same site was also probed using 31P NMR with a phosphorothioate at the A9 position. These techniques provide a signal from a functional metal site whose population modulates ribozyme activity.
Relating metal sites to ribozyme function The advantage of a clear signal from a metal site is that it allows correlation of the population of that particular site with the appearance of ribozyme activity. In the case of the hammerhead ribozyme, knowing the apparent affinity of the A9/G10.1 site allows us to conclude that at least two types of metal sites influence hammerhead activity. This conclusion is reached because, at 1 M NaCl, the A9/G10.1 site (Kd 10 mM) is populated at Mn2þ concentrations much lower than the >1 mM Mn2þ required for full activity. Thus, even though the PS-rescue experiment shows that hammerhead activity is sensitive to the high-affinity metal site, at least one other weaker site(s) is also required. The picture of high- and low-affinity cation interactions being required for ribozyme function has been echoed by other recent studies. Hampel and Burke [12] have very recently compared the cation requirements of hammerhead ribozyme folding and activity. Folding, as measured by solvent protection in a hydroxyl radical footprinting experiment, takes place at cation concentrations signif-
icantly lower than those required for catalysis, with Mg2þ or Liþ as the supporting cation. Interestingly, a similar situation has recently been described for the HDV ribozyme. Using thermal denaturation as a measure of ribozyme folding, Bevilacqua and co-workers [15] propose a structural Mg2þ site(s) (Hill coefficient 1) with an apparent affinity much higher than that of a catalytic Mg2þ interaction. From their work on the folding of RNase P RNA, Fang et al. [9] predict that a final rearrangement from a relatively stable folding intermediate to the active form of the ribozyme involves small structural changes around a specific metal ion site. These studies point to an overall picture of ribozymes in which population of highaffinity cation sites leads to relatively large structural rearrangements. The final fine-tuning of an active site, however, may require population of cation site(s) with relatively weak affinity. Such a situation is depicted schematically in Figure 4. Whether a final ‘fine-tuning’ of ribozyme activities by cations with relatively weak affinities makes the cations ‘catalytic’ cofactors may be a question of nomenclature. At the beginning of this review, ‘catalytic’ metal ions were defined as those that contact groups that change bond order during the chemical reaction. It is certainly the case that thiophilic metal ions that rescue deleterious effects of sulfur substitutions at ribozyme active sites fall within this definition of catalytic cofactors. However, even slightly noninnocent single-atom substitutions such as thiophosphates may be able to change the rate-limiting step in ribozyme kinetics. Hard-core enzymologists may prefer the gold standard of isotope effects, whereby the substitution has no structural effect. Direct spectroscopic probes, as well as a better understanding of the effects on RNA structure of substitutions used in ribozyme studies, will aid future investigations of ribozyme activity.
Figure 4
Ma (tight) structural
Mb (weak) structural or catalytic Current Opinion in Structural Biology
Model of classes of metal sites that influence ribozyme activity. As described in the text, in the hammerhead and HDV ribozymes, evidence has been provided of high-affinity structural metal ion sites (Ma) and weaker-affinity metal sites that influence catalysis (Mb). The weaker-affinity sites may directly coordinate and influence moieties involved in the chemical reaction, or may foster small structural changes near the active site that are critical to ribozyme chemistry. The arrow on the right indicates the cleavage site. Current Opinion in Structural Biology 2003, 13:317–324
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Metal ion binding to catalytic RNA molecules DeRose 323
Conclusions In this review, some current studies on the effects of cations on ribozyme activity have been presented. It has been pointed out that separating cation influences on RNA structure from direct involvement in the chemical step of ribozyme reactions is a significant challenge. For large ribozymes, RNA positions whose substitutions affect metal-dependent activity appear in clusters around the ribozyme active site. This suggests that metals aid in the reactions of large ribozymes by directly contacting reactive groups, although the exact details of such contacts remain to be determined for the native RNAs. In the hammerhead and HDV ribozymes, metal-induced folding requires population of cation sites with higher affinities than those required for ribozyme chemistry, leading to a general picture of at least two classes of cation sites. In the hammerhead ribozyme, at least one metal site can be discretely populated, examined in situ and have a strong influence on activity, suggesting a fine-tuning of ribozyme function by a cation cofactor. It is evident that RNA has evolved more than one method of harnessing electrostatics to influence its function. Finally, it is important to note that, for at least two of the small ribozymes, although cations certainly influence function, the majority of the catalytic power is probably derived from nucleobases [34]. The possible interplay between cation sites and base pKas has been noted [15,25].
Acknowledgements The author wishes to thank the Section editors, Carl C Correll and David MJ Lilley, for helpful comments. Support from the NIH (GM58096), NSF (CHE0111696) and THECB is gratefully acknowledged.
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. Doudna J, Cech T: The chemical repertoire of natural ribozymes. Nature 2002, 418:222-228. A review of the first 20 years of ribozyme investigations. 2. Fedor MJ, Westhof E: Ribozymes: the first 20 years. Mol Cell 2002, 10:703-704. A brief but important account of news from a meeting held in celebration of the 20-year anniversary of ribozymes. 3.
DeRose VJ: Two decades of RNA catalysis. Chem Biol 2002, 9:961-969.
4. Pyle AM: Metal ions in the structure and function of RNA. J Biol Inorg Chem 2002, 7:679-690. This article reviews structural aspects of known metal sites in RNA and their influences on function. It concludes with a review of work that identified a cluster of metal interactions with the D5 helix of the group II intron.
reactions stimulated by monovalent and divalent cations. RNA 2001, 7:537-545. This article, one of a few that characterize the activity of the small ribozymes in high concentrations of monovalent cations, describes a careful comparison of the effect of many different single-atom substitutions on hammerhead ribozyme activity under different ionic conditions. The authors conclude that the majority of substitutions have the same effect for both monovalent and divalent cations, with some interesting exceptions of phosphorothioates at the A9 and cleavage sites, and that therefore the overall mechanism is very similar under very different cation backgrounds. 7.
Nakano S, Proctor DJ, Bevilacqua PC: Mechanistic characterization of the HDV genomic ribozyme: assessing the catalytic and structural contributions of divalent metal ions within a multichannel reaction mechanism. Biochemistry 2001, 40:12022-12038.
8.
Sigel R, Vaidya A, Pyle A: Metal ion binding sites in a group II intron core. Nat Struct Biol 2000, 7:1111-1116.
9.
Fang X-W, Thiyagarajan P, Sosnick TR, Pan T: The rate-limiting step in the folding of a large ribozyme without kinetic traps. Proc Natl Acad Sci USA 2002, 99:8518-8523. In this work, the folding of the RNase P RNA is investigated kinetically and by SAXS techniques. Evidence is provided of a rate-limiting step in the folding pathway that depends on a relatively small, but energetically significant, rearrangement of a metal ion already bound in a previous step. 10. Hammann C, Lilley DMJ: Folding and activity of the hammerhead ribozyme. Chembiochem 2002, 3:690-700. 11. Rangan P, Masquida B, Westhof E, Woodson SA: Assembly of core helices and rapid tertiary folding of a small bacterial group I ribozyme. Proc Natl Acad Sci USA 2003, 100:1574-1579. A recent and characteristically elegant example of time-resolved hydroxyl radical footprinting techniques applied to metal-dependent RNA folding. 12. Hampel KJ, Burke JM: Solvent protection of the hammerhead ribozyme in the ground state: evidence for a cation-assisted conformational change leading to catalysis. Biochemistry 2003, 42:4421-4429. This paper describes a direct comparison of metal-dependent RNA folding, probed by hydroxyl radical footprinting experiments, with the Mg2þ or Liþ requirements for hammerhead ribozyme catalysis. In both cases, the concentration of metal required for RNA folding is significantly lower than that required for catalysis. 13. Russell R, Millettt IS, Tate MW, Kwok LW, Nakatani B, Gruner SM, Mochrie SGJ, Pande V, Doniach S, Herschlag D, Pollack L: Rapid compaction during RNA folding. Proc Natl Acad Sci USA 2002, 99:4266-4271. SAXS provides a global picture of ion-induced folding in the Tetrahymena group I intron ribozyme. 14. Zhuang X, Rief M: Single-molecule folding. Curr Opin Struct Biol 2003, 13:88-97. A recent review that includes the pioneering single-molecule fluorescence studies of folding in the hairpin ribozyme. 15. Nakano S, Cerrone A, Bevilacqua P: Mechanistic characterization of the HDV genomic ribozyme: classifying the catalytic and structural metal ion sites within a multichannel reaction mechanism. Biochemistry 2003, 42:2982-2994. In another careful examination of ribozyme activity as a function of cation background, Nakano et al. established three channels for HDV ribozyme activity that differ slightly in cation influence. Here, by populating different channels, the characteristics of a structural and a ‘catalytic’ metal are established through both activity and thermal denaturation studies. 16. Eckstein F: Developments in RNA chemistry, a personal view. Biochimie 2002, 84:841-848.
5. Misra VK, Draper DE: The linkage between magnesium binding and RNA folding. J Mol Biol 2002, 317:507-521. In this paper and references therein, Misra and Draper lay out a thermodynamic framework for metal interactions with RNA in terms of several different contributions, the balance of which defines apparent affinities for both specific and nonspecific cation sites. The results from Poisson– Boltzmann calculations are in good agreement with experimental parameters.
17. Smith JS, Nikonowicz EP: Phosphorothioate substitution can substantially alter RNA conformation. Biochemistry 2000, 39:5642-5652.
6.
19. Horton TE, Maderia M, DeRose VJ: Impact of phosphorothioate substitutions on the thermodynamic stability of an RNA GAAA
O’Rear JL, Wang SL, Feig AL, Beigelman L, Uhlenbeck OC, Herschlag D: Comparison of the hammerhead cleavage
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18. Maderia M, Horton TE, DeRose VJ: Metal interactions with a GAAA RNA tetraloop characterized by P-31 NMR and phosphorothioate substitutions. Biochemistry 2000, 39:8190-8200.
Current Opinion in Structural Biology 2003, 13:317–324
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tetraloop: an unexpected stabilization. Biochemistry 2000, 39:8201-8207. 20. Maderia M, Hunsicker LM, DeRose VJ: Metal-phosphate interactions in the hammerhead ribozyme observed by P-31 NMR and phosphorothioate substitutions. Biochemistry 2000, 39:12113-12120. 31 P NMR spectroscopy was used to directly probe Cd2þ interactions with phosphorothioate-substituted sites in the hammerhead ribozyme. As expected from metal-rescue experiments, high-affinity coordination to the hammerhead A9 position is observed. Unexpectedly, weak to little interaction with the cleavage site phosphorothioate is detected. 21. Murray J, Dunham C, Scott W: A pH-dependent conformational change, rather than the chemical step, appears to be ratelimiting in the hammerhead ribozyme cleavage reaction. J Mol Biol 2002, 315:121-130. An X-ray crystallographic study of hammerhead ribozyme structures trapped by methods designed to capture kinetic intermediates. The resulting model suggests a small but critical pH-dependent conformational change that precedes phosphate cleavage. 22. Wang S, Karbstein K, Peracchi A, Beigelman L, Herschlag D: Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate. Biochemistry 1999, 73:14363-14378. 23. Suzumura K, Yoshinari K, Tanaka Y, Takagi Y, Kasai Y, Warashina M, Kuwabara T, Orita M, Taira K: A reappraisal, based on 31P NMR, of the direct coordination of a metal ion with the phosphoryl oxygen at the cleavage site of a hammerhead ribozyme. J Am Chem Soc 2002, 124:8230-8236. 24. Tanaka Y, Kojima C, Morita E, Kasai Y, Yamasaki K, Ono A, Kainosho M, Taira K: Identification of the metal ion binding site on an RNA motif from hammerhead ribozymes using 15 N NMR spectroscopy. J Am Chem Soc 2002, 124:4595-4601.
27. Shan S, Kravchuk AV, Piccirilli JA, Herschlag D: Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction. Biochemistry 2001, 40:5161-5171. Kinetic analyses of several different single-atom substitutions around the active site of the group I intron provide evidence of the model shown in Figure 2. Several metal interactions are predicted. 28. Christian EL, Kaye NM, Harris ME: Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA. EMBO J 2002, 21:2253-2262. In this paper, several different phosphorothioate and base substitutions are explored on or near the P4 helix of RNase P RNA, which is thought to create the active site in cleaving the 30 end of tRNA. Evidence of the set of metal ions shown in Figure 2 is presented. 29. Sigel RKO, Vaidya A, Pyle AM: Metal ion binding sites in a group II intron core. Nat Struct Biol 2000, 7:1111-1116. 30. Gordon PM, Sontheimer EJ, Piccirilli JA: Kinetic characterization of the second step of group II intron splicing: role of metal ions and the cleavage site 20 -OH in catalysis. Biochemistry 2000, 39:12939-12952. 31. Morrissey SR, Horton TE, DeRose VJ: Mn2þ sites in the hammerhead ribozyme investigated by EPR and continuouswave Q-band ENDOR spectroscopies. J Am Chem Soc 2000, 122:3473-3481. This is the first description of a metal site using ‘advanced’ EPR techniques to identify RNA ligands. EPR and ENDOR spectroscopy show that a high-affinity Mn2þ site in the hammerhead ribozyme can be populated, exhibits a perturbed EPR signal and can be probed to extract evidence of phosphate and aqua ligands, as well as a nucleobase. 32. Morrissey SR, Horton TE, Grant CV, Hoogstraten CG, Britt RD, DeRose VJ: Mn2þ-nitrogen interactions in RNA probed by electron spin-echo envelope modulation spectroscopy: application to the hammerhead ribozyme. J Am Chem Soc 1999, 121:9215-9218.
25. Huppler A, Nikstad LJ, Allmann AM, Brow DA, Butcher SE: Metal binding and base ionization in the U6 RNA intramolecular stem-loop structure. Nat Struct Biol 2002, 9:431-435.
33. Hoogstraten CG, Grant CV, Horton TE, DeRose VJ, Britt RD: Structural analysis of metal ion ligation to nucleotides and nucleic acids using pulsed EPR spectroscopy. J Am Chem Soc 2002, 124:834-842.
26. Yean SL, Wuenschell G, Termini J, Lin RJ: Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome. Nature 2000, 408:881-884.
34. Bevilacqua PC: Mechanistic considerations for general acidbase catalysis by RNA: revisiting the mechanism of the hairpin ribozyme. Biochemistry 2003, 42:2259-2265.
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