INTRODUCTION

The catalytic cleavage of an RNA phosphodiester bond to a 2,3-cyclic phosphate by the hammerhead ribozyme requires the participation of divalent metal ions. McKay and co-workers (1) observed a single bound metal ion when Mn²⁺ or Cd²⁺ was soaked into hammerhead crystals, with the metal ion in close proximity to the pro-R_p-oxygen of the P9 phosphate and N₇ of the guanine base at position 10.1 (Fig. 1). Previous biochemical experiments suggested that both of these groups were important in hammerhead cleavage: the P9 pro-R_p-oxygen was identified in phosphorothioate interference experiments (2, 3), and a role for a purine at position 10.1 was implicated in nucleotide substitution experiments (4, 5). More recently, the N₇ of this purine was implicated by the ability of guanine, but not 7-deazaguanine, to efficiently rescue the activity of a ribozyme with an abasic nucleotide at position 10.1 (6).

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These results, taken together, support a model in which binding of a metal ion to the P9 pro-R_p-oxygen and the N₇ of G10.1 affects catalysis. However, the metal ion site identified in the crystal structure is ~20 Å from the cleavage site phosphodiester, with no obvious connection to this site. In addition, there are no data directly demonstrating a functional role for the structurally identified metal ion, nor are there quantitative data that indicate how important this metal ion might be for catalysis. Finally, coordination of a metal ion to the pro-S_p-oxygen of P9 rather than the pro-R_p-oxygen was suggested from crystallographic data with a different ribozyme construct (7).

We have therefore tested this model and quantitated the functional consequences of perturbing this site by substituting the pro-R_p- and pro-S_p-phosphoryl oxygen atoms at position P9 with sulfur and following catalysis in the presence and absence of Cd²⁺, a thiophilic metal ion. The results provide strong support for the model and indicate that a metal ion coordinated at the pro-R_p position is critical for efficient catalysis.

EXPERIMENTAL PROCEDURES

Materials

The ribozymes and substrates were prepared by solid phase synthesis (8). Variants of each ribozyme containing a phosphorothioate in position 9 were produced by published sulfurization methods (9), which result in a nearly racemic mixture of R_P and S_P diastereomers (10). For HH1 (Scheme 1), the two isomers of the P9 phosphorothioate (referred to as thio-P9_Rp and thio-P9_Sp) were separated by reverse phase HPLC¹ (11). When each 5-³²P-labeled oligonucleotide was digested with snake venom phosphodiesterase, a 12-nucleotide species accumulated, consistent with a phosphorothioate at position 9. Furthermore, the 12-nucleotide species from the second HPLC peak was cleaved more slowly than that from the first peak, suggesting that the second peak is the S_P-phosphorothioate isomer (12). For HH16 (Scheme 1), the ribozyme length (38 nucleotides) prevented efficient large scale separation of the thio-isomers.

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Substrates were 5-end-labeled using [-³²P]ATP and T4 polynucleotide kinase and purified by nondenaturing polyacrylamide gel electrophoresis. Oligonucleotide concentrations were determined using specific activities for radioactive RNAs and assuming a residue extinction coefficient of 8.5 × 10³ M¹ for nonradioactive RNAs.

MgCl₂ and CdCl₂ (>99.99%) were purchased from Aldrich. Buffers were from Sigma (molecular biology grade). CdCl₂ solutions were used immediately after preparation or were made as concentrated, acidic stocks (pH 2) and diluted into buffer immediately prior to use.

Methods

All reactions were single turnover and were carried out with ribozyme in excess of 5-end-labeled substrate at 25 °C, in 50 mM buffer (pH 6.5) (PIPES·Na for experiments with HH1 and BisTris-propane·HCl for experiments with HH16) and 10 mM MgCl₂, unless otherwise indicated. The reaction protocols were essentially as described previously (13, 14). Ribozyme and substrate were annealed prior to initiating reactions by the addition of divalent metal ions. Control reactions varying the final concentration of ribozyme indicated that the substrate was completely bound in all cases. Data were fit to the appropriate kinetic equation using KaleidaGraph (Synergy Software) or SigmaPlot (Jandel Scientific) and gave fits with R² > 0.99 in all cases. Values of k₂ varied <25% between independent experiments.

Reactions of the HH16 construct containing a phosphorothioate in position 9 yielded biphasic reaction time courses, with each phase corresponding to about half of the total reaction. These time courses were fit to the sum of two independent exponentials, giving independent k₂ values for each phase. The fast phase, characterized by a k₂ value nearly identical to that of the unmodified ribozyme, was attributed to the thio-P9_Sp isomer, and the slower phase was attributed to the thio-P9_Rp isomer based on the results with the resolved HH1 thio-isomers. The rates and relative amplitudes of the two phases did not change when the annealed ribozyme-substrate complex was diluted by 150-fold (decreasing the ribozyme concentration from 600 to 4 nM) immediately after starting the reaction, arguing against kinetic complexities arising from multimeric ribozyme complexes. Also, the reaction time course did not change when the ribozyme-substrate complex was diluted and chased at the start of the reaction with a large molar excess of an HH16 variant (with G5 replaced by an abasic residue) that binds substrate normally but does not react (6).² This suggests that there is no dissociation of the pre-annealed ribozyme-substrate complex even over the longest time courses (48-96 h). Each phase of the time course was ~10-fold faster at pH 7.5 than at pH 6.5, as expected if each process were limited by the chemical step (15). Finally, purification of this phosphorothioate-substituted HH16 by anion exchange HPLC (8) resulted in partial separation of ribozyme forms such that the two phases had identical rate constants to those observed in the racemic mixture but different relative amplitudes (one fraction gave 0.8 of the fast component and 0.2 of the slow, whereas a second fraction gave 0.2 of the fast and 0.8 of the slow).

Rates and relative amplitudes of the two phases for reactions in 10 mM Mg²⁺ did not change upon addition of 0.2 mM EDTA or 2 mM dithiothreitol to the reaction mixture, suggesting that neither kinetic process depended on the presence of contaminating metal ions. In reactions with added Cd²⁺, the concentration of EDTA carried over from the ribozyme and substrate stocks was <15 nM.

RESULTS

We have used two different hammerhead ribozyme constructs, HH1 and HH16 (Scheme 1), in testing the role and importance of the metal ion identified in the x-ray crystallographic structure. Each of these ribozymes is kinetically and thermodynamically well characterized (13, 14), allowing the chemical cleavage step to be followed. In addition to the added confidence provided by obtaining parallel results with different constructs, specific attributes of each ribozyme were exploited in the experiments described below.

The R_P- and S_P-phosphorothioate isomers at position 9 formed during solid phase synthesis of HH1 (referred to as thio-P9_Rp and thio-P9_Sp ribozyme, respectively) could be separated by HPLC to give fractions with high enrichment of each isomer. In single turnover reactions with saturating ribozyme, cleavage by the thio-P9_Sp ribozyme proceeded at the same rate, within error, as that of the unmodified ribozyme (k₂ = 0.7 ± 0.1 min¹; 10 mM Mg²⁺, 25 °C (pH 6.5)). In contrast, catalysis by the thio-P9_Rp ribozyme was substantially reduced, with an observed cleavage rate constant of k₂ = 0.028 min¹. This supports the previous qualitative observations of compromised ribozyme function upon substitution of an R_P-phosphorothioate at position P9 (2, 3) and is consistent with a functional interaction with the pro-R_p-, but not pro-S_p-, oxygen at position P9.

The observed rate decrease of 25-fold upon substitution of the P9 pro-R_P-oxygen of HH1 with sulfur represents a lower limit for the effect of this change on the chemical step. This limit arises because the ribozyme preparation could contain a small amount of phosphate or S_P-phosphorothioate contaminant. Because dissociation of bound substrate from HH1 is fast on the time scale of the reaction (k_off^S = 0.4 min¹; (14)), the substrate can exchange between different ribozyme molecules, even in a single turnover experiment performed with saturating concentrations of ribozyme. A thio-P9_Rp preparation contaminated with only 4% of unmodified or thio-P9_Sp ribozyme would show a 25-fold rate decrease, even if the thio-P9_Rp ribozyme was completely inactive. Consistent with this possibility, lowering the temperature to slow the exchange of substrate between different ribozymes gave a much larger observed effect from the thio-P9_Rp substitution (500-fold at 4 °C; data not shown).

To circumvent this problem, we determined the thio effect using a different hammerhead construct, HH16 (Scheme 1). Substrate dissociation is immeasurably slow for this ribozyme, with a calculated t1/2 of ~10,000 years (13). Because exchange does not occur on the time scale of the reaction, each substrate molecule is cleaved by whichever ribozyme it initially binds, the thio-P9_Sp or thio-P9_Rp. Single turnover substrate cleavage should therefore occur in two independent phases, each corresponding to the reaction of one isomer population. As expected, two separate kinetic phases, each corresponding to reaction of about half of the substrate, were observed (Fig. 2). The first phase occurs at essentially the same rate as the wild type reaction (Table I) and was assigned as reaction of the thio-P9_Sp ribozyme, based on the results with the defined isomers of HH1 described above. The slow phase was similarly assigned as reaction of the thio-P9_Rp ribozyme. (Control experiments supporting this interpretation are described under "Methods.") The cleavage rate for the slow, thio-P9_Rp isomer is 500-fold slower than that for the unmodified ribozyme (Table I). Thus, the effect of this single atom substitution is much larger than was determined using HH1 under the same conditions. Indeed, it was this paradoxical result that led us to propose the fast exchange model for HH1 and test it at low temperature, as described above. The results indicate that catalysis by both ribozymes is greatly compromised by this thio substitution.

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Table I. Effect of phosphorothioate substitution at position 9 on the hammerhead cleavage reaction in the presence and absence of Cd2+ Single-turnover reactions with HH16 in 50 mM BisTris-propane·HCl (pH 6.5), 25 °C. Metal ions k2 Unmodified Thio-P9Sp Thio-P9Rp min1 10 mM Mg2+ 0.07 0.06 1.4 × 104 10 mM Mg2+ + 100 µM Cd2+ 0.4 0.3 1.2 Ratio 6 5 8500

The large deleterious effect of the R_P-phosphorothioate at position 9 was obtained in reactions carried out in the presence of Mg²⁺, which is a "hard" divalent metal ion having a low affinity for sulfur (16-18). To determine if the slow reaction of the thio-P9_Rp ribozyme resulted from loss of a metal ion bound at this site, reactions were carried out with low concentrations of Cd²⁺, a strongly thiophilic metal ion, and with 10 mM Mg²⁺ present to minimize the effect of Cd²⁺ at other sites. As shown in Table I, the addition of 100 µM Cd²⁺ increased the cleavage by the thio-P9_Rp ribozyme over 10⁴-fold, while having only ~5-fold effects on the unmodified and thio-A9_Sp ribozymes. In addition, in the presence of Cd²⁺, the thio-P9_Rp ribozyme reacts at nearly the same rate as the unmodified and thio-P9_Sp ribozymes. Thus, the "rescue" is complete. Similar results were obtained with HH1 (data not shown). Analogous metal ion rescue has been observed with protein enzymes and other ribozymes (e.g. Refs. 16 and 19-24). The ability to observe efficient rescue strongly suggests that the deleterious effect from the P9 R_P-phosphorothioate arose from disruption of a Mg²⁺ site and indicates that this metal ion is of critical importance for hammerhead function.³

The dependence of the cleavage rate for the thio-P9_Rp and wild type ribozyme was measured as a function of [Cd²⁺] to determine the apparent metal ion affinity for the P9 site (Fig. 3A). The marked dependence for the thio-P9_Rp ribozyme and the shallow dependence for the wild type and thio-P9_Sp ribozymes (Fig. 3A and data not shown) indicate that this large effect is specific for the P9 R_P-thio-isomer. The Cd²⁺ concentration dependence of k_2(obs) for the thio-P9_Rp ribozyme suggests that binding of a single Cd²⁺, which has an equilibrium constant for dissociation from the ribozyme-substrate complex of K_d = 25 µM, is responsible for increasing the activity by ~10⁴-fold.⁴

Cd

Cd

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The association of this metal ion and its rate effects are summarized in Fig. 3B. Transition states can be considered as if they were species in equilibrium with ground states, according to transition state theory. This allows the Cd²⁺ affinity of the transition state to be calculated: the 10⁴-fold faster reaction with Cd²⁺ bound indicates that Cd²⁺ binds 10⁴-fold stronger to the transition state than to the ground state, corresponding to a dissociation constant, K_d, of 2.5 nM.

DISCUSSION

The functional importance of a distinct metal ion observed in the x-ray crystallographic structure of the hammerhead ribozyme (Fig. 1A; Ref. 1) has been tested. There is a large deleterious effect from substituting the pro-R_p-phosphoryl oxygen at position 9 with sulfur for reactions carried out in Mg²⁺ alone, and small amounts of Cd²⁺, a thiophilic metal ion, restore the activity to unmodified levels. These observations provide strong support for an important functional role of this metal ion. Consistent with this interpretation, modification of G10.1, which contains the other ligand observed in the structure, decreases the ability of Cd²⁺ to rescue the deleterious effect of the thio substitution at P9.⁵

The rate decreases by 10³-fold upon replacing the P9 pro-R_p-oxygen with sulfur when Mg²⁺ is the only divalent metal ion. This corresponds to a loss of 4 kcal/mol in transition state stabilization upon loss of a bound metal ion,⁶ rivaling the effects from nucleotide substitution or excision of bases from the conserved hammerhead core (6, 28).

The P9 metal ion plays a critical role in hammerhead catalysis, despite its large distance from the labile phosphodiester group in the ground state. The ground state Cd²⁺ affinity of 25 µM is similar to that for adenosine 5-O-thiomonophosphate (K_d = 24 µM (18)). The properties of this metal binding site change dramatically during catalysis. In the transition state, there is a 10⁴-fold increase in Cd²⁺ affinity, relative to the ground state (Fig. 3B), corresponding to an additional 5.3 kcal/mol of binding free energy. This large increase suggests that there is at least one additional Cd²⁺ ligand in the transition state. For comparison, the additional carboxylate ligand of nitrilotriacetate relative to iminodiacetate increases Cd²⁺ affinity by 5.6 kcal/mol (K_d = 10^9.5 and 10^5.4 M, respectively (29)). An alternative model in which the Cd²⁺ ligands are better positioned in the transition state than in the ground state cannot be ruled out; however, this model would require the observed low nanomolar transition state affinity to be achieved with coordination by only two ligands, the sulfur at P9 and N₇ at G10.1.

How can this metal ion, which is ~20 Å from the reactive phosphoryl group in the hammerhead crystal structures (1, 7, 30), exert such a large effect, and what could an additional ligand(s) be? McKay pointed out that the ground state complex observed by crystallography would have to rearrange prior to cleavage to allow an in-line attack and also noted that the observed structure and structural variants with modest conformational rearrangements could not readily account for catalytic interactions or for roles of substituents that had been shown to be functionally important (1, 28). For example, the base of G5 is critical for catalysis, yet it engages in no interactions with the rest of the ribozyme (Fig. 1A). These observations suggest that a large scale conformational rearrangement may be required prior to cleavage. Such a conformational rearrangement could allow formation of additional transition state interaction(s) of the metal ion at P9 and could account for the importance of this metal ion in catalysis.⁷

We present the following speculative model for this conformational transition as a starting point for future discussions. We suggest that domain I rearranges and docks onto the major groove face of domain II. Several functional groups that are important for catalysis are located on the major groove face of domain II. In addition, the widened major groove face of this domain includes the pro-R_p-oxygens 5 of P13 and P14, which are important in the transition state (3)⁸ but appear to lack contacts in the ground state structure. The substantial network of interactions in domain II and the maintenance of the metal ion binding site at P9 and G10.1 in the transition state are consistent with domain II remaining largely unaltered in the transition state and serving as a "receptor" for domain I. A substantial rearrangement of domain I upon docking could account for the critical catalytic importance of functional groups such as those on G5 that do not make extensive ground state interactions. Finally, we suggest that the core is more packed in the active conformation with extensive interconnections between the conserved residues, consistent with the deleterious effects from removal of individual bases or 2-hydroxyl groups that are large relative to overall catalysis (6, 28).⁹

In summary, a large scale conformational rearrangement may be required for the hammerhead to adopt its catalytic conformation. Subsequent to this conformational change, does the P9 metal ion interact directly at the cleavage site or does it exert its effect indirectly through the folded structure? Establishing the identity of the additional ligand to the metal ion bound at P9 would provide a test of the proposed model and would provide an important constraint for the active conformation of the hammerhead ribozyme.