Functional Identification of Catalytic Metal Ion Binding Sites within RNA

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-S P phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.


Introduction
Phosphoryl transfer reactions occur ubiquitously in biology, playing roles in gene replication, recombination, and expression.To orchestrate these central biological processes, biomacromolecules have harnessed the catalytic power of divalent metal ions [1][2][3][4][5][6][7][8][9][10].The chemistry underlying these cellular events hinges critically on the metal ion interactions that occur during function.However, there exists little functional data defining the coordination environment of individual metal ions during catalysis and correspondingly few methods for obtaining such information.
Structural analyses can serve as a powerful starting point for investigation of catalytic metal ions, but these approaches provide no direct information about transition-state interactions.Reflecting this uncertainty, for a given enzyme or enzyme family the number of active-site metal ions and their proposed interactions during catalysis can vary with both enzyme and observer ([2-10] and references therein).For example, restriction enzymes have been crystallized with zero, one, two, or three metal ions in their active sites [7,8,10], and different RNase H crystal structures support either a one or two metal ion mechanism [3].This variability in metal ion binding also occurs for RNA.In structures of the hammerhead ribozyme and tRNA, Mg 2þ and Mn 2þ occupy different sites [11,12], and recent group I intron structures exhibit differences in the number, location, and identity of active-site metal ions [13][14][15].Thus, only the combination of structural and functional studies can establish metalloenzyme mechanisms unambiguously.
To understand how metalloenzymes utilize the catalytic power of metal ions, we must identify individual catalytic metal ions functionally, determine their relationships to each other, define their coordination environment, and establish the network of interactions that position the coordinating groups.Metal ion rescue experiments using substrate mutations offer a strategy to identify specific catalytic metal ion interactions within enzyme active sites, a particularly formidable challenge in ribozymes due to the sea of metal ions that interact electrostatically with the anionic phosphodiester backbone.Functionally deleterious sulfur or nitrogen perturbations that exhibit rescue upon increasing cation softness (e.g., replacing Mg 2þ with Cd 2þ or Mn 2þ ) suggest direct metal ion coordination during catalysis [16,17].This approach has revealed catalytic metal ion interactions with enzyme substrates in the Tetrahymena group I ribozyme [18][19][20][21], the ai5c group II intron [22,23], RNase P [24], the hammerhead ribozyme [25,26], the human spliceosome [27,28], and many protein enzymes (e.g., [16,[29][30][31][32] and references therein).
The Tetrahymena group I ribozyme catalyzes nucleotidyl transfer from an oligonucleotide substrate that mimics the natural 59-splice site to an exogenous guanosine (G) that serves as the nucleophile in a reaction analogous to the first step of group I intron self-splicing (Equation 1) [33,34].
within the oligonucleotide substrate and G nucleophile that interact with metal ions in the chemical transition state [18][19][20][21].To determine whether one or several distinct metal ions mediate these interactions, Shan et al. developed thermodynamic fingerprint analysis, quantitatively analyzing the reactivity of modified substrates relative to unmodified substrates over a range of rescuing metal ion concentrations [35].In this approach, the reactions for both modified and native substrates start from the same ground state and monitor the same elementary reaction steps.The resulting rescue profiles serve as distinctive ''fingerprints'' for the rescuing metal ion(s), revealing by comparison whether the same or distinct metal ions interact with the identified substrate ligands.Thermodynamic fingerprint analysis and related analyses [36] using a series of substrates bearing single or multiple atomic perturbations have provided functional evidence for a network of three distinct metal ions within the Tetrahymena ribozyme active site (Figure 1), making a total of five interactions with the reaction's transition state.Metal ions coordinate to the 39-oxygen leaving group (M A ), the 39oxygen on the G nucleophile (M B ), and the 29-hydroxyl of the G nucleophile (M C ).Two of these metal ions (M A and M C ) also contact the pro-S P oxygen of the scissile phosphate [35,36].However, the ligands within the ribozyme core architecture that bind and position these metal ion cofactors remain largely unknown, leaving the catalytic metal ion coordination environments undefined.
The non-bridging phosphate oxygens of the RNA backbone commonly serve as ligands for divalent metal ions.For the Tetrahymena group I ribozyme and other RNA enzymes, phosphorothioate interference studies have generated a plethora of ligand candidates for metal ions [17,26,[37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52].However, there have been few attempts to link these putative ligands to metal ions directly involved in catalysis [42,53,54].Using the Tetrahymena group I ribozyme as a model system, we have combined thermodynamic fingerprint analysis with an array of atomically perturbed substrates and ribozyme siteand stereo-specific phosphorothioate mutations to develop a general functional approach for identifying ligands for the catalytic metal ions.Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

Results
Choosing Sites for Phosphorothioate Substitution within the Ribozyme Core Backbone mutation sites were chosen prior to the release of the recently reported group I intron structures [13][14][15].To guide our choice of substitution sites, we focused on previously reported interferences arising from random R Pphosphorothioate incorporation into the phylogenetically conserved core regions of the Tetrahymena group I intron.As Mg 2þ coordinates poorly to sulfur, the R P -phosphorothioate interferences could reflect direct disruption of a metal ion interaction with the pro-R P phosphate oxygen, indirect disruption of a metal interaction with the geminal pro-S P phosphate oxygen, or other effects.A literature survey identified 14 sites of R P -phosphorothioate interference within the ribozyme's conserved core [17,47,52,55].Herein we analyze ribozymes containing site-specific phosphorothioate incorporation at six sites within the J6/7 and P7 regions of the ribozyme (Figure 2A).We constructed these mutant ribozymes semi-synthetically with both R P -and S Pphosphorothioate mutations at these six sites (Figure 2B), resulting in 12 variant ribozymes.
The variant ribozymes were characterized kinetically within the known framework of the Tetrahymena ribozyme reaction (Figure 3; [33,56,57] and references therein).The oligonucleotide substrate (S; Table 1) binds to the ribozyme (E) in two steps.First, S forms Watson-Crick base pairs with the ribozyme's internal guide sequence (see Figure 2A) to give the open complex (E Á S) O .The resulting P1 helix then ''docks'' into the ribozyme core via tertiary interactions, forming the closed complex (E Á S) C ([33,57-59] and references therein).G binds to give the ternary (E Á S Á G) C complex, and the reaction proceeds through the phosphoryl transfer step (k chem ), resulting in cleavage of the oligonucleotide substrate.
We first tested whether Cd 2þ , a thiophilic metal ion that can adopt octahedral coordination geometry like Mg 2þ [60][61][62], stimulates the ability of the phosphorothioate containing ribozymes to catalyze oligonucleotide substrate cleavage (Figure 4).Under conditions of saturating ribozyme and G  [35,36].The 29-OH of U(-1) participates in a hydrogenbonding network with the 29-OH of A207 and the exocyclic amine of the G Á U wobble pair [58,72] and donates a hydrogen bond to the adjacent 39-oxygen in the transition state [71]; the hydrogen bonds are shown as hashed lines.DOI: 10.1371/journal.pbio.0030277.g001(10 mM MgCl 2 ), several of the phosphorothioates affected catalysis significantly (data not shown, and see Table 2 below), but upon addition of 0.1-1.0 mM Cd 2þ , only one of the variant ribozymes, the C262-S P variant, experienced significant stimulation (Figure 4 and data not shown).This stimulation suggested that a functionally important Cd 2þ phosphorothioate interaction may occur at the C262-S P position, leading us to focus predominantly on this variant ribozyme.The other positions tested, in principle, remain viable ligand candidates as the absence of rescue cannot be taken as evidence that the modification site does not serve as a metal ion ligand.However, metal ion rescue experiments analogous to those described below for the C262-S P ribozyme suggest that none of the other phosphorothioate substitutions have large effects on binding of the known catalytic metal ions (see Figure S1).

Cd 2þ Accelerates a Non-Chemical Step in the C262-S P Ribozyme Reaction
Metal ion rescue experiments provide definitive information about transition-state metal ion-ligand interactions only when conducted under conditions in which the same reaction steps are monitored and the chemical step limits the reaction rate [35].To learn more about the apparent Cd 2þ stimulation and to establish appropriate reaction conditions under which the chemical step could be monitored, we undertook basic characterization of the C262-S P ribozyme.We first present data concerning the rate-limiting step of the C262-S P ribozyme reaction, and then assess the effects of this mutation on individual reaction steps.
Previous work established that, in the wild-type (WT) ribozyme, the chemical step follows a pre-equilibrium loss of a proton, presumably from the 39-hydroxyl of the attacking G (see Figure 1).This leads to a log-linear pH dependence with a slope of one under conditions of rate-limiting chemistry [33,[63][64][65].However, in the absence of Cd 2þ , the pH dependence for cleavage of the À1d,rSA 5 substrate (Table 1) by the C262-S P variant ribozyme is essentially flat above pH 5 (Figure 5A, closed circles), in contrast to the slope of one for the WT ribozyme [63,65].This difference suggests that, under the reaction conditions, a conformational change limits the rate for the variant ribozyme.In the presence of low concentrations of Cd 2þ , the variant behaves more like the WT ribozyme, exhibiting a log-linear pH dependence with slope one (Figure 5A, open circles).This Cd 2þ -induced change in pH dependence suggests that Cd 2þ accelerates the nonchemical step sufficiently to render the chemical step ratelimiting throughout the entire pH range.
Although the molecular basis for the rate-limiting, nonchemical step and its stimulation by Cd 2þ in the C262-S P ribozyme reaction remains undefined, comparison to the reaction of À1r,dSA 5 offers some insight.The À1r,dSA 5 substrate reacts from the ternary complex (E Á S Á G) with a log-linear pH dependence whose slope approximates one, even in the absence of Cd 2þ (Figure 5B, closed circles).Addition of Cd 2þ has no effect on the reaction rate or the pHdependence (Figure 5B, open circles).These results suggest that the chemical step limits the reaction of À1r,dSA 5 both in the presence and absence of Cd 2þ .AsÀ1r,dSA 5 binds primarily in the open complex with the WT ribozyme (see Figure 3) The ribozyme conserved core is highlighted in blue, and the six positions of phosphorothioate substitution within the J6/7 and P7 regions are labeled in green.The internal guide sequence at the 59-terminus of the ribozyme is labeled ''IGS.''(B) Ligation strategy for constructing mutant ribozymes.Following ion exchange HPLC purification of the phosphorothioate diastereomers (C), the phosphorothioate-substituted mutation oligonucleotides were ligated into full-length ribozymes by two successive-splint mediated ligations using T4 DNA ligase [77], as described in Materials and Methods.DOI: 10.1371/journal.pbio.0030277.g002[36,66] and À1d,rSA 5 binds in the closed complex [57,58], the rate-limiting non-chemical step observed with À1d,rSA 5 may reflect phosphorothioate-induced formation of an altered closed complex that must rearrange prior to the chemical step.Acceleration of this non-chemical step depends on Cd 2þ ; increasing the Mg 2þ concentration does not accelerate this step, nor does Mn 2þ or Zn 2þ (data not shown).The Cd 2þ dependence suggests that this non-chemical step may require metal ion coordination to the backbone phosphorothioate.Future study of this metal-ion-dependent non-chemical step revealed by phosphorothioate incorporation may provide an opportunity for deeper understanding of the role of metal ion coordination in conformational rearrangements within RNA.
This initial characterization of the C262-S P ribozyme allowed us to choose appropriate reaction conditions for further characterization.For analyzing the binding of G and oligonucleotide substrates to the variant ribozyme, we included Cd 2þ in sufficient but subsaturating concentrations to render the chemical step rate-limiting without influencing the measured binding constants (i.e., subsaturating Cd 2þ in order to have no significant effect on substrate affinities; [Cd 2þ ] 1 mM) [35].For metal ion rescue reactions in which a constant Cd 2þ concentration was not feasible, we chose oligonucleotide substrates (Table 1) or reaction pHs that render the chemical step rate-limiting.

Phosphorothioate Substitution Effects on Reactivity and Binding
We analyzed the 12 phosphorothioate-substituted variants within the J6/7 and P7 regions of the ribozyme (see Figure 2A) under a variety of reaction conditions.As noted above, reactions with the C262-S P variant contained Cd 2þ to maintain rate-limiting chemistry.Cd 2þ had no significant effect on the behavior of the other ribozymes (data not shown).Representative data from this survey-phosphorothioate substitution effects on the reaction E Á S Á G ! products (k ternary ) and G binding to the E Á S complex ðK G dðEÁSÞ Þ-are given in Table 2.This survey revealed one position at which phosphorothioate substitution strongly perturbs (greater than 10-fold) G binding: the S P phosphorothioate at nucleotide C262, the same variant that exhibited Cd 2þ stimulation.
Based on the G-binding results, the Cd 2þ rescue of a nonchemical step, and screens for the effects of J6/7 and P7 phosphorothioates on rescue by the catalytic metal ions (see Figure S1), we decided to characterize the C262-S P ribozyme further, focusing on how the phosphorothioate affects the individual reaction steps (see Figure 3).We found that the C262-S P substitution had no significant effect on oligonucleotide substrate association or dissociation (Table S1).We then further investigated the observed effect of the C262-S P phosphorothioate substitution on G binding.In reactions with the À(1-3)d,rSA 5 substrate, which binds to the WT ribozyme in the open complex (E Á S) O (based on observed G- binding affinity [56,67] and Mn 2þ stimulation of cleavage activity [66] [data not shown]), G binds to the C262-S P variant and the WT ribozyme with the same affinity.Thus, in the absence of docked oligonucleotide substrate, the C262-S P phosphorothioate has no effect on G binding (Table 3).The C262-S P phosphorothioate affects subsequent reaction steps, however.The ternary open complex, (E Á S Á G) O , for the variant reacts about 40-fold slower than that for the WT ribozyme, suggesting possible effects on oligonucleotide substrate docking into the G-bound active site or on the chemical step.
In the presence of WT ribozyme, G and S bind cooperatively to give the ternary closed complex, (E Á S Á G) C .G binds to (E Á S) C five- to ten-fold tighter than to (E Á S) O or free enzyme [56,67].Using À1d,rSA 5 to form (E Á S) C and À(1-3)d,rSA 5 to form (E Á S) O , we reproduced this coupling for the WT ribozyme (Table 3).In contrast, the C262-S P ribozyme exhibited no coupling.Indeed, the presence of docked oligonucleotide substrate with the C262-S P ribozyme weakens G binding.Overall, the C262-S P ribozyme closed complex has an approximately 40-fold weaker affinity for G than does the WT ribozyme closed complex (Table 3).Reaction chemistry from the ternary closed complex remains unaffected, as   The ribozyme binds the oligonucleotide substrate (in two steps) and the exogenous G that serves as the nucleophile in the ribozyme reaction as described in the text ( [33,56,57] and references therein).
and K G d(E Á S) c are G dissociation constants from free E, (E Á S) O , and (E Á S) C , respectively.K IGS d is the dissociation constant for the oligonucleotide substrate from the internal guide sequence, and K dock and K9 dock are the docking equilibria for the E Á S and E Á S Á G complexes, respectively.K S d(E) and K S d(E Á G) are the observed dissociation constants for the oligonucleo- tide substrate from free E and the E Á G complex, respectively, and k chem is the observed rate of oligonucleotide substrate cleavage.DOI: 10.1371/journal.pbio.0030277.g003 (E Á S Á G) C formed with À1d,rSA 5 has the same rate for the WT and C262-S P ribozymes (Table 3).
Previous work has linked the coupled binding of G and the oligonucleotide substrate to M C , which mediates contacts to the G 29-hydroxyl and the scissile phosphate pro-S P oxygen (see Figure 1) [68,69].The loss of coupled binding between the oligonucleotide substrate and G induced upon C262-S P phosphorothioate substitution therefore raised the possibility that the C262 pro-S P phosphoryl oxygen resides near the M C binding site in the ribozyme tertiary structure.

A Linkage between C262-S P and One of the Catalytic Metal Ions
The apparent functional connection between the C262 pro-S P phosphoryl oxygen atom and the metal ion bound at site C (M C ) could occur through direct interaction of this oxygen with M C, or indirectly through a chain of interactions.We used the C262-S P ribozyme to ascertain whether direct coordination occurs, analyzing the metal ion rescue behavior for each of the previously identified metal ion sites.If the C262-S P phosphorothioate directly coordinates to a Cd 2þ ion bound at one of the known catalytic metal ion binding sites, we expect the stronger Cd 2þ -sulfur interaction to shift the metal ion rescue profile toward lower Cd 2þ concentration relative to the WT ribozyme.
We determined the M A , M B , and M C profiles for the WT and C262-S P ribozymes according to approaches described previously [35,36,68].Table 4 lists the substrates and kinetic regimes for the reactions used to obtain each rescue profile.For each rescue profile, WT and mutant ribozyme reactions with both modified and unmodified substrates must start from the same ground state and monitor the chemical step.Unless indicated otherwise, we chose substrates and conditions so that the substrate bearing the modification is not bound within the active site in the starting ground state.This ensures that substrate modifications have no effect on ground-state binding of the rescuing metal ion to the WT  8) leads to log-linear pH dependence (slope ¼ 1.1 6 0.1) over the pH range of 4-7, consistent with the chemical step being ratelimiting [33,[63][64][65].The Cd 2þ rescue observed in Figure 3 results from acceleration of the non-chemical step, as indicated by the vertical arrow, such that this non-chemical step is no longer rate-limiting.The increased slope observed at low pH for reactions both with and without Cd 2þ is consistent with ribozyme inhibition due to multiple independent protonations [65].(B) Cleavage of À1r,dSA 5 under saturating ribozyme and G conditions.Cleavage of À1r,dSA 5 is log-linearly dependent on pH with and without added Cd 2þ (0 mM Cd 2þ , ; 1 mM Cd 2þ , 8 ) (best fit slopes of 0.8 in both cases), consistent with the chemical step being rate-limiting in both cases.DOI: 10.1371/journal.pbio.0030277.g0051) in the presence or absence of 0.1 mM Cd 2þ (10 mM Mg 2þ background).The largest Cd 2þ stimulation occurred with the C262-S P variant ribozyme.Significant Cd 2þ stimulation remained unique to this ribozyme at all Cd 2þ concentrations tested (0.1-10 mM; data not shown).Concentrations of Cd 2þ above 1 mM inhibit the ribozyme reaction with a steep concentration dependence (see, e.g., Figures S4  and S5A).The small Cd 2þ stimulation observed for the U258 variant ribozymes is consistent with coordination of a metal ion important for structural stability, as proposed by Lindqvist et al. [78].DOI: 10.1371/journal.pbio.0030277.g004and mutant ribozymes.The supporting information provides full details of the methods, models, and equations used to fit the Cd 2þ rescue dependencies.
To monitor Cd 2þ binding at the metal ion site A, we followed the reactivity of an oligonucleotide substrate containing a 39-thiophosphoryl linkage at the cleavage site, S m39S (Figure 6A) [21,35]; i.e., Cd 2þ specifically rescues the cleavage rate of S m39S relative to the unmodified 39-oxygen oligonucleotide substrate (k rel , k 39S /k 39O ).The C262-S P and WT ribozymes exhibit nearly identical profiles for rescue at metal ion site A (Figure 6A).The small deviation at high Cd 2þ is beyond experimental error (as indicated by error bars in Figure 6A) and may reflect an indirect effect from Cd 2þ occupancy at a different site (see Protocol S1 ).The lack of significant change in Cd 2þ rescue at metal site A provides no evidence for direct contact between the S P -phosphorothioate at residue 262 and a Cd 2þ ion binding at metal site A. Therefore, we conclude that the C262 pro-S P phosphoryl oxygen in the WT ribozyme is not a ligand for M A .
To follow Cd 2þ binding to the M B site, we monitored Cd 2þ rescue of the reactivity of CUCG 39S A relative to CUCGA in the reverse reaction with E Á P (Equation 2) [20,35].CUCG 39S A contains a 39-thiophosphoryl linkage at the cleavage site.
In reactions catalyzed by both the WT and C262-S P ribozymes, Cd 2þ specifically stimulates the reactivity of CUCG 39S A more than 500-fold.This rescue fits well to a model in which a single Cd 2þ ion binds to the M B site and rescues the reaction (Figure 6B).As C262-S P phosphorothioate incorporation exhibits no effect on the M B Cd 2þ rescue profile, we conclude that the C262 pro-S P phosphoryl oxygen in the WT ribozyme is not a ligand for M B .
To follow Cd 2þ binding to the M C site, we monitored Cd 2þ rescue of oligonucleotide substrate cleavage by 29-aminoguanosine (G N ) relative to G [19,35,68].We first probed Cd 2þ binding to the M C site in the E Á S complex, conducting reactions at subsaturating G or G N concentrations ((k c /K m ) G or (k c /K m Þ GN conditions).Specific Cd 2þ stimulation of cleavage with G N occurs with both the WT and C262-S P ribozymes, giving linear rescue curves throughout the Cd 2þ concentration range tested with slopes that reflect a single Cd 2þ stimulating cleavage by G N (Figure 6C).In contrast to the M A and M B rescue profiles discussed above, the M C rescue curve for the C262-S P variant shifts to the left, with 16-fold lower Cd 2þ concentrations required to achieve the same level of rescue as the WT.This shift suggests that the S P -phosphor- 39S denotes a bridging 39-thiophosphoryl linkage, and PS-Sp and PS-Rp denote SP and RP phosphorothioate linkages, respectively.29-methoxy substitutions were incorporated to prevent miscleavage, as described in Table 1.All residues are 29-OH and all cleavage sites are unmodified phosphodiester linkages unless denoted otherwise.b Control substrates with natural oxygen linkages were chosen so that they bind in the same ground state as sulfur-containing substrates, thereby allowing for correction of non-specific Cd 2þ effects on the ribozyme reactions, as described in the text [35,36].c CUCGA and CUCG39SA were used in MB assays in place of GA and G3'SA, respectively, to enhance binding to the ribozyme through P9.0 base-pairing interactions [56,80].DOI: 10.1371/journal.pbio.0030277.t004Cd 2þ rescue profiles for M A , M B , and M C with WT ( 8 ) and C262-S P (n) ribozymes are displayed.In each case, the specific metal ion-substrate contact being probed is indicated by closed red dots.
(A) M A rescue of S m39S cleavage.In the Tetrahymena ribozyme transition-state model, the oligonucleotide substrate 39-thiophosphoryl modification at U(-1) is shown in green.M A rescue reactions monitoring cleavage of À(1-3d)rSA 5 and S m39S were carried out under E Á S Á G ! products (k ternary ) conditions as described in Materials and Methods.The Cd 2þ profiles for M A rescue are fit to a Hill equation, facilitating comparison between the WT and C262-S P ribozyme profiles (see Protocol S1).The fits give Hill constants of 1 and 1.2 for the WT and C262-S P ribozymes, respectively.Error bars not seen are obscured by the data symbols.
(B) M B rescue of CUCG 39S A cleavage.In the transition-state model, the G 39-thio modification is shown in green.M B rescue reactions monitoring cleavage of CUCGA and CUCG 39S A were performed under E Á P þ CUCG 39X A ! products [(k c /K m ) CUCG(39X)A ] conditions, as described in Materials and Methods.M B rescue is fit to a model in which one Cd 2þ ion binds to E Á P and stimulates reaction of CUCG 39S A. othioate at nucleotide C262 interacts directly with the Cd 2þ ion binding at the M C site, a model we test further below.
The M C rescue profiles for both the WT and C262-S P ribozymes increase linearly up to the highest experimentally accessible Cd 2þ concentrations (Figure 6C), indicating that the rescuing Cd 2þ ion binds to the E Á S complexes of these ribozymes under these conditions with a dissociation constant that exceeds 10 mM.Without saturation behavior, we cannot definitively ascertain whether the C262 phosphorothioate-induced shift in the M C rescue profile emanates from tighter Cd 2þ binding.Consequently, we probed Cd 2þ binding to the M C site in the presence of saturating G N .Bound G N provides M C with an additional ligand, the nitrogen of the 29-amino group, which should interact strongly with Cd 2þ [70].
We conducted the rescue experiment as described above but included G N at saturating concentration to form the E Á S Á G N ternary complex.As with subsaturating G N , both the WT and C262-S P ribozymes experienced significant specific Cd 2þ rescue (Figure 6D).The rescue profile for the WT E Á S Á G N complex remains linear throughout the Cd 2þ concentration range tested, indicating that even in the presence of bound G N , Cd 2þ binds to the M C site with an apparent affinity of greater than 10 mM (50 mM Mg 2þ background).In striking contrast, the Cd 2þ rescue profile for reaction of the C262-S P E Á S Á G N complex exhibits saturation behavior, with an apparent dissociation constant of K Cd d ; 0.2 mM.The C262-S P E Á S Á G N ternary complex therefore binds the rescuing Cd 2þ ion more than 50-fold tighter than does C262-S P ribozyme E Á S binary complex, suggesting that the 29- amino group of G N interacts with M C in the ground state.
Moreover, the C262-S P E Á S Á G N complex binds the rescuing Cd 2þ ion more than 50-fold tighter than does the corresponding WT ternary complex, showing that the phosphorothioate substitution at C262 enhances Cd 2þ binding to metal ion site C. Taken together, these data further support a model in which the C262-S P phosphorothioate interacts directly with the Cd 2þ ion in the M C site and strongly implicate the C262 pro-S P oxygen as a ligand for M C in the WT ribozyme.
An Independent Test of the C262 pro-S P Phosphoryl Oxygen as a M C Ligand The reaction of an oligonucleotide substrate bearing a S Pphosphorothioate at the cleavage site (S Sp , Table 1) also experiences Cd 2þ stimulation with the WT ribozyme.The Cd 2þ rescue profile for this modified oligonucleotide substrate exhibits a slope of two, suggesting that two Cd 2þ ions contact the non-bridging sulfur atom in the transition state [36].Thermodynamic fingerprint analysis established that these rescuing metal ions bind at sites A and C (Figure 7A) [36].The S Sp oligonucleotide substrate therefore offers another strategy by which to test whether the C262-S P phosphorothioate enhances Cd 2þ binding to the M C site.If the M C site in the C262-S P ribozyme becomes saturable in the presence of bound G N , as described in the previous section, then the rescue profile for S Sp cleavage should transition from an apparent dependence on two Cd 2þ ions to a dependence on one Cd 2þ ion.The results described below meet this prediction, thereby providing additional quantitative support for the assignment of the pro-S P phosphoryl atom of residue C262 as a ligand for M C .
The Cd 2þ rescue profile for S Sp cleavage by C262-S P ribozyme with saturating G fits well to a model with two Cd 2þ ions rescuing S Sp cleavage with neither metal ion saturating, analogous to that for the WT ribozyme (Figure 7B) [36].This profile matches expected rescue behavior, given that neither the M A nor M C (subsaturating G N ) individual rescue profiles exhibit saturation with the C262-S P ribozyme (see Figures 6A and 6C, respectively).In contrast, with saturating G N , Cd 2þ rescue of S Sp cleavage by the C262-S P ribozyme no longer fits well to a dependence on two nonsaturating Cd 2þ ions (see Figure 7C and the dashed line in Figure 7D).Rather, the best fit gives a dependence on only one Cd 2þ ion, suggesting that one of the two metal ion sites involved in rescuing the S Sp reaction saturates.Assuming that one Cd 2þ ion binds to the C262-S P ribozyme with K Mc;app d ¼ 0.2 mM, the value determined for Cd 2þ binding to metal site C in the presence of saturating G N (see Figure 6D), we obtain the solid line in Figure 7D.In contrast, for the WT ribozyme, Cd 2þ rescue of the S Sp reaction in the presence of saturating G N exhibits a dependence on two Cd 2þ ions up to the highest accessible Cd 2þ concentration [36].Therefore, as predicted, the C262-S P phosphorothioate substitution enhances binding of one of the rescuing Cd 2þ ions in the reaction of S Sp , strongly supporting the conclusion that M C interacts directly with the pro-S P atom of the C262 phosphate.

Discussion
Metalloenzymes that catalyze phosphoryl transfer play multiple roles throughout biology, serving as kinases, phosphatases, polymerases, and nucleases, among other functions [1][2][3][4][5][6][7][8][9][10].We have established a powerful new experimental paradigm with which to identify ligands that coordinate to catalytic metal ions.Our analysis of simultaneous atomic perturbations within the Tetrahymena ribozyme core and its substrates, under conditions that allow valid thermodynamic comparisons, provides strong evidence that the pro-S P oxygen at residue C262 serves as a ligand for metal ion C (M C , Figure 8A).A single phosphorothioate substitution at this site, alone and in combination with G N , changes the metal ion affinity and specificity at the M C site while having little or no effect on the M A and M B sites.Although we cannot determine unambiguously whether the interaction between the pro-S P oxygen at C262 and M C occurs via outer sphere or inner sphere coordination in the natural ribozyme, the presence of a direct, inner sphere metal ion-ligand interaction provides the simplest model to account for our observations.More extensive rearrangement by the ribozyme active site to accommodate the backbone mutation appears less likely, as the metal ion that contacts C262-S P satisfies all the transitionstate contacts proposed for M C .Further demonstrating the efficacy of this approach, detailed analysis of phosphorothioate mutations in the P4 and J5/4 regions of the ribozyme core identified the pro-S P oxygen at position C208 as a ligand for M A (AVK, JLH, JAP, and DH, unpublished data), consistent with the previous proposal of Szewczak et al. [53].
Under some conditions, the C262-S P ribozyme E Á S Á G ternary complex must undergo a rate-limiting conformational change en route to the chemical transition state.Cd 2þ accelerates this non-chemical step in this C262-S P ribozyme, suggesting that M C , which interacts with the phosphorothioate during the reaction, mediates this conformational change.Currently, we lack sufficient information to speculate further on the molecular basis of this conformational change.The ability to induce a new or existing conformational change may provide a future opportunity to investigate the relationship between RNA function and dynamics.
Crystal structures of three different group I introns have emerged in the past several months, providing a structural context for these functionally identified catalytic metal ligands [13][14][15].The three structures converge beautifully with respect to the global architecture and reveal the highly electrostatic character of the active site, consistent with functional studies.These structures also underscore the difficulty of defining catalytic metal ion interactions, exhibiting limited agreement regarding the active-site conformation and the number, charge, and location of bound metal ions.This variability in metal ion number and location notwithstanding, the C262 pro-S P oxygen (or its equivalent) lies in proximity to the xG 29-hydroxyl in all three structures (Figure 8).The Azoarcus and Tetrahymena crystals contain electron density for a metal ion within coordination distance of this phosphoryl oxygen [14,15], whereas the Twort ribozyme structure lacks such electron density [13].The data presented herein establish the importance of this proximity for function and suggest that the active-site configuration in the crystals bears at least some relevance to the active-site configuration in the transition state.This agreement between structural and functional data for metal ion C supports the assignment of a conserved catalytic metal ion binding site at the top of the P7 helix in group I introns (see Figure 2A).
Extensive group I intron biochemical investigations, conducted for two decades in the absence of atomic resolution structures, have revealed an intricate network of interactions surrounding the reaction center in the transition state interconnected by hydrogen bonds, metal ion coordinations, and the atomic configuration of the reactants (see Figure 1).The results suggest a constellation of three metal ions at the active site [35,36], making five atomic interactions to the transition state.In addition to these catalytic metal ion coordinations, the 29-hydroxyl group of U(-1) donates a hydrogen bond to the adjacent 39-oxygen leaving group in the transition state [71].The 29-hydroxyl group of A207 donates a hydrogen bond to the 29-OH of U(-1) and accepts a hydrogen bond from the exocyclic amine of G22, thereby bridging the cleavage site 29-OH and the G Á U wobble pair [58,72].The recent group I intron crystal structures, though unable to define the number, location, or catalytic interactions of metal ions unambiguously, provide an opportunity to visualize how these functionally defined networks extend deeper into the ribozyme core.
Based on the recent crystal structure of the Azoarcus group I intron, an alternative model for the transition state was proposed in which only two metal ions interact with the reaction center [15].In this model, one metal ion coordinates to the 39-oxygen leaving group of the cleavage site uridine and the pro-S P oxygen of the scissile phosphate, the same interactions proposed for M A in the functional model (Figure 8A).The other metal ion coordinates directly with the 29hydroxyl group of G as does M C in the functional model, but coordinates indirectly to the pro-Sp oxygen of the scissile phosphate, in contrast to the direct interaction implicated by the functional data.A metal ion interaction with the 39oxygen of G, mediated by M B in the functional model, is absent from the crystallographic model.No direct evidence for a M B -binding site was obtained from the X-ray structure.The resting structure of the RNA in the crystal could adopt a conformation that differs from the active structure and thereby exclude M B from the crystals.Alternatively, the M B rescue profile could reflect the recruitment of a thiophilic metal ion to the active site during the reaction of the sulfurcontaining G analogue.As the 39-oxygen of the G undergoes a large charge rearrangement in the transition state, we suggest that a direct metal ion interaction with the 39-oxygen of G is likely, such that M B is present in the normal reaction or metal ion C makes both 29-and 39-interactions with G [73].These different proposals for specific metal ion coordination configurations during the ribozyme reaction highlight the need for further structural and functional tests and refinements of the catalytic models.
To achieve a unified description of catalytic function that integrates the biochemically defined interaction networks and the crystallographically defined three-dimensional structure, we must establish ''anchor points''-functionally verifiable linkages between transition-state interactions and the enzyme's core.Our analysis establishes the C262 pro-S P oxygen as such an anchor point.Anchor points provide critical information about the spatial arrangement of catalytic groups within the global architecture of the enzyme.Together, the transition state model, anchor points, and the recent X-ray structures establish a powerful foundation to build toward an in-depth understanding of how cooperative structure adopted by this RNA and other enzymes engenders enormous catalytic power and exquisite specificity.

Materials and Methods
Materials.WT Tetrahymena ribozyme was prepared as described previously [34].All oligonucleotide substrates (see Table 1) were prepared and 59-end-labeled using standard methods [34,35,54,74].Oligonucleotides with thio substitutions were prepared by published procedures [75].Oligonucleotides containing phosphorothioate diastereomers were separated by anion exchange HPLC [42,54].Reverse phase HPLC of purified diastereomers, under conditions in which the R P diastereomer elutes before the S P , allowed assignment of each diastereomer's configuration [76].
Ribozyme preparation.Variant ribozymes were constructed semisynthetically using successive splint-mediated ligations [77].Synthetic oligonucleotides corresponding to nucleotides 255-274 of the ribozyme, each containing a single phosphorothioate modification at the desired mutation site, were purchased from Dharmacon (Lafayette, Colorado, United States).Following phosphorothioate separation, synthetic oligonucleotides were 59-phosphorylated with T4 polynucleotide kinase and cold ATP.Constructs corresponding to nucleotides 22-254 and 274-409 of the Tetrahymena ribozyme were transcribed using DNA templates produced by PCR truncation of the plasmid-encoded ribozyme sequence, with excess GMP present in the transcription of the 39-construct to yield a 59-monophosphate.The transcripts were ligated to the synthetic oligonucleotide via two successive splint-mediated ligations with T4 DNA ligase to yield full-length ribozyme containing a single R P -or S P -phosphorothioate mutation at the desired site.
Ligated ribozymes appear to contain approximately 40% inactive enzyme fraction, as indicated by biphasic kinetics under conditions in which oligonucleotide substrate cleavage occurs faster than oligonucleotide substrate dissociation (data not shown).Evidence suggests that the inactive ribozyme fraction in these semisynthetic ribozymes may be due to errors at the ligation junctions resulting from ligation of a subset of transcribed RNA constructs with incorrect termini (K.Travers, V. Diankov, and DH, unpublished data).Under conditions used in this work to characterize variant ribozyme reactivity, the inactive fraction did not contribute to the cleavage activity monitored in our assays of ribozyme activity.The inactive fraction did not affect association and dissociation rate constants measured by pulse chase experiments, as evidenced by monophasic binding behavior (data not shown).The activity of WT ribozyme constructed by ligation varied less than 2-fold from that of transcribed WT ribozyme (Table S2) after accounting for the presence of the inactive ribozyme fraction.The inactive ribozyme fraction does not affect the conclusions in this work.
General kinetic methods.All cleavage reactions were single turnover, with ribozyme in excess of radiolabeled S (S*), and were carried out at 30 8C in 50 mM buffer and 50 mM MgCl 2 unless noted otherwise.All reactions without Cd 2þ contained 0.1 mM EDTA.The buffers used were NaOAC (pH 4.0-5.2),NaMES (pH 5.6-6.7), and NaMOPS (pH 7.0-7.5).Reaction mixtures containing all components except Cd 2þ , EDTA, and radiolabeled oligonucleotide substrate were pre-incubated at 50 8C for 30 min to renature the ribozyme.Reactions were followed and analyzed as described previously [33,36,74].
Determination of rate and equilibrium constants.For oligonucleotide substrates that bind to the WT ribozyme in the closed complex, e.g.À1d,rSA 5 (see Table 1), we define k c as the first-order rate constant for the reaction of the ternary complex (E Á S Á G) C ! products.Values of k c were determined at pH 7.0, with ribozyme saturating with respect to oligonucleotide substrate (20-100 nM E, K S d , 1 nM) and with saturating G (2 mM, K G dðEÁSÞ as reported in Table 2).
For cleavage of oligonucleotide substrates that are known to bind in the open complex to the WT ribozyme, e.g., À1r,dSA 5 and S Sp [36,66], we define k ternary as the first-order rate constant for the  2 and 3, and data not shown).(kc/Km) G and (k c /K m Þ GN are the second-order rate constants for the reaction E Á S þ G (or G N ) ! products.In these experiments, we used the À1r,dSA 5 oligonucleotide substrate that binds to the WT and variant ribozymes in the open complex [36,66].Values of (k c /K m ) G and (k c /K m Þ GN were determined at pH 7.0, with ribozyme saturating with respect to oligonucleotide substrate   ; 100-150 lM) ( [68], Table 3, and data not shown).
(k c /K m ) CUCG(39X)A (where X ¼ O or S) is the second-order rate constant for the reaction E Á P þ CUCGA !products.Values of (k c / K m ) CUCG(39X)A were determined at pH 6.5 with trace amounts of radiolabeled CUCG 39X A and E Á P subsaturating with respect to CUCG 39X A (X ¼ O, 10-20 nM E Á P, K CUCGA d .100 nM; X ¼ S, 100-200 nM E Á P, K CUCGð39SÞA d .400 nM) (data not shown).To maintain the chemical step as rate-limiting in reactions catalyzed by the WT ribozyme, an oligonucleotide product with a 29-deoxyribothymidine at the 39-terminus was used (CCCUCdT); 29-deoxyribose incorporation at this site slows the chemical step approximately 10 3 -fold [74].Reactions in the presence of the C262-S P variant ribozyme used an all-ribose oligonucleotide product (CCCUCU).
Association (k on ) and dissociation (k off ) rate constants were measured by a gel mobility shift assay using pulse-chase methods [33,56].Specific conditions and experimental details for these experiments are described in Protocol S2.
Experimental errors.All titrations, binding constants, and rate determinations were repeated at least three times.Error bars, when present, indicate standard deviation from at least three determinations.Error bars may be obscured by data symbols.Plots without error bars display representative data.Reported errors in all tables, both in the text and Supporting Information, are the standard deviation of at least three independent measurements.Data analysis.The Cd 2þ concentration dependencies for rescue of reaction of modified substrates were analyzed according to previously described methods ( [35,36] and Protocol S1).

Figure 1 .
Figure 1.Model of the Tetrahymena Ribozyme Transition State during the First Step of Splicing The three identified catalytic metal ions (M A , M B , and M c ) and their transition-state interactions with the U(-1) 39-oxygen, G nucleophile 39-oxygen, and G 29-OH, respectively, along with the M A and M C interactions with the scissile phosphate pro-S P oxygen, are shown by red dots [35,36].The 29-OH of U(-1) participates in a hydrogenbonding network with the 29-OH of A207 and the exocyclic amine of

Figure 2 .
Figure 2. Construction of Ribozymes Containing Site-Specific Phosphorothioate Substitutions (A) Secondary structure of the Tetrahymena group I ribozyme.The ribozyme conserved core is highlighted in blue, and the six positions of phosphorothioate substitution within the J6/7 and P7 regions are labeled in green.The internal guide sequence at the 59-terminus of the ribozyme is labeled ''IGS.''(B) Ligation strategy for constructing mutant ribozymes.Following ion exchange HPLC purification of the phosphorothioate diastereomers (C), the phosphorothioate-substituted mutation oligonucleotides were ligated into full-length ribozymes by two successive-splint mediated ligations using T4 DNA ligase [77], as described in Materials and Methods.DOI: 10.1371/journal.pbio.0030277.g002 39-O,P-O rA rA rA rA rA -(1-3)d,rSA 5 rC rC rC dU dC dU 39-O,P-O rA rA rA rA rA -1r,dSA 5 dC dC dC dU dC rU 39-O,P-O rA dA dA dA dA S m39S b mC mC mC rU rC dU 39-S,P-O rA S Sp b mC mC mC rU rC dU 39-O,P-S rA CUCGA rC rU rC rG 39-O,P-O rA CUCG 39S A r C r U r C r G 39-S,P-O rA a r ¼ 29-OH; d ¼ 29-H; m ¼ 29-OCH3; 39-S and 39-O indicate the presence of a sulfur or oxygen atom as the 39-bridging atom of S, respectively; P-S and P-O indicate the presence of a sulfur or oxygen as the SP or pro-SP atom of the scissile phosphate of S, respectively.b 29-OCH3 groups were introduced into the (-4-6) positions of Sm39S and SSp to prevent miscleavage and simplifyanalysis of substrate cleavage reaction kinetics, as reported previously[57,58].These substitutions have no effect on kinetic or thermodynamic reaction parameters[57,58].DOI: 10.1371/journal.pbio.0030277.t001

Figure 3 .
Figure 3.The Tetrahymena Ribozyme Reaction PathwayThe ribozyme binds the oligonucleotide substrate (in two steps) and the exogenous G that serves as the nucleophile in the ribozyme reaction as described in the text ([33,56,57] and references therein).K G d(E) , K G

Figure 5 .
Figure 5.The pH Dependence of C262-S P Ribozyme Activity Reveals a Non-Chemical Rate-Limiting Step (A) Cleavage of À1d,rSA 5 under saturating conditions, as given in Figure 3.In the absence of Cd 2þ ( Á ), the pH dependence flattens above pH ;4.7; the data are fit to k max ¼ 1/(1þ10 n(pKa-pH) ).Addition of 1 mM Cd 2þ (8) leads to log-linear pH dependence (slope ¼ 1.1 6 0.1) over

Figure 4 .
Figure 4.The Effect of Cd 2þ on Activity of Phosphorothioate-Containing Ribozymes Reactions contained saturating G and ribozyme, and monitored cleavage of À1d,rSA 5 (see Table1) in the presence or absence of 0.1 mM Cd 2þ (10 mM Mg 2þ background).The largest Cd 2þ stimulation occurred with the C262-S P variant ribozyme.Significant Cd 2þ stimulation remained unique to this ribozyme at all Cd 2þ concentrations tested (0.1-10 mM; data not shown).Concentrations of Cd 2þ above 1 mM inhibit the ribozyme reaction with a steep concentration dependence (see, e.g., FiguresS4 and S5A).The small Cd 2þ stimulation observed for the U258 variant ribozymes is consistent with coordination of a metal ion important for structural stability, as proposed by Lindqvist et al.[78].DOI: 10.1371/journal.pbio.0030277.g004 ) was followed as a function of G concentration.Reactions were performed with saturating ribozyme (100 nM, no change in observed cleavage rates in reaction with 20 nM ribozyme), 10 mM MgCl2, 50 mM NaMOPS, pH 7.0, at 30 8C. a kternary is the rate constant for EÁSÁG !products, as described in Materials and Methods.b Reactions using the C262-SP ribozyme contained 0.1 mM CdCl2.DOI: 10.1371/journal.pbio.0030277.t002

Figure 6 .
Figure 6.The C262-S P Phosphorothioate Perturbs the Rescue Profile of M C .
(C) M C rescue of À1r,dSA 5 cleavage by subsaturating G N ((k c /K m ) G(or GN) conditions).In the transition-state model, the 29-amino group of the G nucleophile is shown in blue.M C rescue of À1r,dSA 5 cleavage under E Á S þ G(or G N ) ! products ((k c /K m ) G(or GN) conditions) was performed as described in Materials and Methods.k rel data were fit to a model in which one Cd 2þ ion binds and rescues reaction with G N .(D) M C rescue of À1r,dSA 5 cleavage by saturating G N .In the transition-state model, the 29-amino group of the G nucleophile is again shown in blue.M C rescue of À1r,dSA 5 cleavage under E Á S Á G/G N !products (k ternary ) conditions was measured as described in Materials and Methods.k rel data were fit to a model in which one Cd 2þ ion binds and rescues reaction with G N ; with the C262-S P ribozyme, the fit to this model gives K Mc d,app ¼ 0.22 6 0.08 mM.Cd 2þ dependencies of observed cleavage rates in the M A , M B , and M C rescue reactions are displayed in Figures S2, S3, and S4, respectively.DOI: 10.1371/journal.pbio.0030277.g006

Figure 7 .
Figure 7. Cd 2þ Rescue of S Sp Cleavage by the C262-S P Ribozyme Reactions were performed under E Á S Á G/G N !products (k ternary ) conditions, as described in Materials and Methods, with À(1-3)d,rSA 5 serving as the unmodified control oligonucleotide substrate.Cd 2þ dependencies for oligonucleotide substrate cleavage by G and G N are displayed in Figure S5.(A) Model of the Tetrahymena ribozyme transition state for rescue of S Sp reaction by Cd 2þ ions bound at M A and M c .Closed red circles indicate metal ion coordinations considered important for rescue, and the S P phosphorothioate modification at the scissile phosphate is denoted in green.(B) k rel versus Cd 2þ for cleavage of S Sp with G.The data are fit to a model in which two Cd 2þ ions rescue reaction, with neither Cd 2þ binding site saturating (see Protocol S1).(C) Model of the Tetrahymena ribozyme transition state for rescue of S Sp cleavage by two Cd 2þ ions bound at M A and M C in the presence of saturating G N .Metal ion coordinations important for rescue are denoted by closed red circles, and the G N amino group is shown in blue.(D) k rel versus Cd 2þ for cleavage of S Sp with G N .The dotted line is a fit to the model used in (B), for two Cd 2þ ions rescuing reaction and neither Cd 2þ binding site saturating.The solid line is a fit to a model in which two Cd 2þ ions rescue reaction, with one site saturating with a K Mc d,app ¼ 0.2 mM (see Protocol S1).DOI: 10.1371/journal.pbio.0030277.g007

Figure 8 .
Figure 8. Functional and Structural Models of Group I Intron Active Sites (A) Model of the Tetrahymena ribozyme transition state from functional data with the C262 pro-S P phosphoryl oxygen and C208 pro-S P oxygen coordinating to M C and M A , respectively ([35,36,53,58,71,72, 74], data herein, and AVK, JLH, JAP, and DH, unpublished results).(B) Model of Mg 2þ binding in the crystal structure of a thermostable variant of the Tetrahymena group I ribozyme (derived from PDB file 1X8W) [14].The model shown is derived from molecule C, although the position of the metal ion appears to vary among the four molecules observed in the asymmetric unit.The putative Mg 2þ ion (dashed green circle) is 2.4 A ˚from the pro-S P oxygen of C262 and 2.1 A ˚from the terminal G (xG) 29-OH.U(-1) is not shown, as the crystallized form of the thermostable Tetrahymena variant ribozyme lacks this nucleotide.(C) Model of K þ binding from the crystal structure of the Azoarcus group I ribozyme (derived from PDB file 1T42) [15].The putative K þ ion (dashed green circle) is 2.4 A ˚from the pro-S P oxygen of G128 (C262 homologue) and 2.8 A ˚from the modeled position for the terminal G (xG) 29-OH; the Azoarcus intron construct that was crystallized contained 29-deoxyguanosine at xG, and electron density for the K þ ion is observed only in the presence of this 29deoxyguanosine modification at xG. (D) Model of proposed Mg 2þ binding site derived from the crystal structure of the Twort group I ribozyme [13].The crystallographic data lack clear density for a metal ion in the region expected for the M C binding site but show the pro-S P phosphoryl oxygen of A120 (C262 homologue) and the 29-OH of xG appropriately juxtaposed to coordinate a single Mg 2þ ion (purple circle).DOI: 10.1371/journal.pbio.0030277.g008 nM E, ribozyme concentration at least 4-fold above K S d , data not shown) and with subsaturating G or G N (30 lM G or G N , K G dðEÁSÞ O ; 500 lM; K GN dðEÁSÞ O

Table 1 .
Oligonucleotide Substrates Used Herein

Table 2 .
G Binding and Reactivity for Ribozymes with Site-Specific Phosphorothioate Substitutions within J6/7 and P7 k ternary (min À1 ) a k rel Cleavage of À1d,rSA5 (see Table1

Table 3 .
G Binding and Reactivity with WT and C262-S P Ribozymes

Table 4 .
Metal Rescue Assays Used to Probe Catalytic Metal Ions in the Tetrahymena Ribozyme