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Nucleic Acids Research Pages 479-484  

A re-investigation of the thio effect at the hammerhead cleavage site
Introduction
Materials And Methods
   RNA synthesis
   Metal ion stock solutions
   Kinetics
Results
Discussion
Acknowledgement
References

A re-investigation of the thio effect at the hammerhead cleavage site

A re-investigation of the thio effect at the hammerhead cleavage site

Edmund C. Scott and Olke C. Uhlenbeck*

Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA

Received October 5, 1998; Revised and Accepted November 20, 1998

ABSTRACT

The effect of introducing a phosphorothioate at the hammerhead cleavage site was investigated using a kinetically well-characterized hammerhead. In buffers containing Mg ion, the RP-phosphorothioate isomer cleaved 2000- to 80 000-fold slower than the SP isomer or the unmodified RNA substrate. Addition of low concentrations of several thiophilic metal ions, especially Cd2+, to these reactions is sufficient to fully restore the cleavage rate of the RP substrate without affecting cleavage rate of the all-oxygen or SP substrate. Thus, a model proposing coordination of a divalent metal ion to the pro-R oxygen at the hammerhead cleavage site appears justified.

INTRODUCTION

The hammerhead ribozyme is a small catalytic RNA molecule that catalyzes the cleavage of its backbone at a specific phosphodiester bond to generate products with 2[prime],3[prime]-cyclic phosphate and 5[prime]-hydroxyl termini (1,2). The reaction proceeds at a rate of ~1/min in millimolar concentrations of divalent metal ion, typically Mg2+. When the hammerhead is prepared with a phosphorothioate linkage at the scissle phosphodiester bond, the cleavage rate is drastically altered, but only for the RP isomer (3). The SP isomer cleaves well and proceeds with inversion of configuration about the phosphorothioate bond (4-6). Although the cleavage rates of the RP isomers reported in the previous studies vary, it is clear that the rate is reduced by more than a factor of 30. Studies of phosphorothioate containing dinucleotides, mononucleotides and uridine 3[prime]-aryl derivatives show that there is no intrinsic difference in the reactivity of a phosphate versus a phosphorothioate linkage (7-10). Therefore, the slow rate of cleavage of the RP isomer reflects disruption of the catalytic mechanism, similar to several protein enzymes catalyzing phosphoryl transfer reactions (reviewed in 11).

An important observation is that the cleavage rate of a hammerhead containing an RP-phosphorothioate at the cleavage site is substantially increased in the presence of Mn2+ ion (3,4,6,12). Such a `manganese rescue' phenomenon can be explained by the relative preference for different divalent metal ions to bind sulfur and oxygen. Experiments using nucleotide mono-, di- and tri-phosphates demonstrate that Mg2+ binds poorly to sulfur, whereas Mn2+ binds better (13,14). Restoration of hammerhead activity in Mn2+ led to a model proposing that Mg2+ binds to the pro-RP oxygen at the cleavage site (3). This model is consistent with the observation that a crystal of an all-RNA hammerhead frozen shortly after the addition of Mg2+ showed a Mg2+ ion bound to pro-RP oxygen of the cleavage site phosphate (15).

While restoration of hammerhead activity by manganese has been known for several years, a re-investigation was needed to address several concerns. The hammerhead substrate (and possibly the ribozyme) used for the original experiments folds into several stable alternate conformations (16). As a result, the Km for this hammerhead is high (2.3 µM) meaning the original experiments were performed under subsaturating conditions (3). Thus, there is uncertainty as to the magnitude of the thio effect, as well as the step in the reaction pathway that is affected. Also, the original experiments were performed in Tris-HCl (pH 7.5) at varying concentrations of Mn2+, with or without 0.5 mM spermine. These reaction conditions can potentially complicate interpretation of the results in several ways. First, since Mg2+ and Mn2+ differ in their propensity to bind the oxygen and nitrogen ligands present in RNA (17), a hammerhead in Mn2+ may fold slightly differently than a hammerhead in Mg2+, and thus have different cleavage properties that are unrelated to the reaction occurring at the cleavage site phosphate. Secondly, at the pH used in the previous experiments, the Mn2+ concentration approached or exceeded its solubility limit (18). Finally, polyamines and Tris buffer bind Mg2+ and Mn2+ differently, also potentially complicating the interpretation of the experiment (19).

Recent work has questioned whether the Mn2+ rescue phenomenon is due to Mn2+ binding to the RP-phosphorothioate at all (20). The authors propose that the effects of Mn2+ on the cleavage rate of thio-substituted hammerheads, derive solely from the lower pKa of water molecules bound to Mn2+, and not from the propensity of Mn2+ to bind sulfur. Their conclusion is based on the observation that, under certain conditions, replacing Mg2+ with Mn2+ increases the cleavage rate of an unmodified hammerhead by as much as the corresponding thio-modified hammerhead. A subsequent mechanistic proposal for hammerhead catalysis therefore did not include a metal ion coordinated to the pro-R oxygen of the cleavage site phosphate (21). In this paper, we re-investigate the rescue experiment using a kinetically well-characterized hammerhead under saturating conditions. A high concentration of Mg2+ or Ca2+ together with a much lower concentration of several different thiophilic metal ions were used. The reaction buffer was changed to ensure complete solubility of the metal ion and omit components that would compete for metal ion binding.

MATERIALS AND METHODS

RNA synthesis

Ribozyme 8 and substrate 8 (Fig. 1) were made by in vitro transcription and chemical synthesis, respectively, as described by Fedor and Uhlenbeck (22). The phosphorothioate linkage was introduced into the substrate molecule during chemical synthesis using the Beaucage reagent (3H-1,2-benzodithiole-3-one 1,1-dioxide) (23). Separation of RP and SP isomers of the thio-modified substrate was achieved by reverse phase HPLC (Waters 60F pump with 600E controller, and 400E UV/visible detector) on a C18 column (Beckman, ultrasphere, C18, 10 mm × 25 cm, semi-preparative) with the following buffers and buffer gradient: 0-5 min, 100% buffer A (0.1 M ammonium acetate, pH 6.8); 5-10 min, 95% buffer A, 5% buffer B (20% buffer A, 80% acetonitrile); 10-90 min, 89% buffer A, 11% buffer B; 90-120 min, column wash of 100% buffer B followed by 100% buffer A. The first peak eluted at 64.2 min and the second at 67.8 min. The leading peak was designated RP due to its lower retention time (11). The identity of each isomer was also consistent with a hammerhead cleavage performed in previous studies (3,4,6,12) in which the RP isomer cleaves more slowly than the SP isomer. The phosphorothioate containing RNAs were stored in filter sterilized, distilled and deionized water at -20°C. No degradation due to repeated freezing and thawing was observed.

   (A)
   (B)

Figure 1. HH8 and phosphorothioate isomers. (A) HH8 is comprised of a 12 nt substrate (open letters) and a 34 nt ribozyme. The arrow indicates the cleavage site where the phosphorothioate linkages were incorporated. (B) The RP- and SP-phosphorothioate isomers.

Metal ion stock solutions

Stock solutions of 2 M MgCl2 and CaCl2 were prepared by dissolving the salt in water through which argon had been bubbled for 1 h to displace the dissolved CO2, and by adding concentrated nitric acid to a final concentration of 100 mM. The completed stock solutions were filter sterilized using a 0.2 µm cellulose acetate membrane. The HNO3 ensured that the pH of the stock solution remained low and the metal ions were completely solubilized. Stocks of 0.1 M MnCl2, CoCl2 and CdCl2 were similarly prepared. The presence of HNO3 in the metal stocks was not enough to effect the pH of the well-buffered reactions after dilution to reach the final metal ion concentrations used. All metal ions were stored at -20°C and thawed for use in the reaction buffers. The metal-chloride salts were the purest grade available from Aldrich Chemical Co. (Milwaukee, WI) and contained the following trace impurities: MgCl2 [(99.995%) Na 2 p.p.m.]; CaCl2 [(99.99%) Pb 60 p.p.m.]; MnCl2 [(99.99%) Pb 35 p.p.m., Co 3 p.p.m.]; CoCl2 [(99.999%) no trace impurities]; CdCl2 [(99.99%) Au 20 p.p.m.].

Kinetics

Single turnover reactions were performed as described previously (22) with minor changes. Aliquots of 50 mM MOPS and PIPES buffers were used at pH 6.5-7.0. The RNAs were heated to 95°C for 1 min in buffer without metal ions in order to dissociate aggregates formed upon freezing. Following cooling of the ribozyme to room temperature, both the ribozyme and substrate strands were incubated briefly in separate tubes with MgCl2 and the appropriate thiophilic metal. Reactions were initiated by adding ribozyme solution to substrate solution. Reaction rates at saturating ribozyme concentrations varied by <30% when the reaction rate exceeded 0.01/min. All values are the averages of at least two determinations.

In order to measure the very slow cleavage rate of RPS8, 40 µl reactions were prepared containing 50 mM MOPS (pH 7.0), 1.5 or 0.5 µM ribozyme, and trace (<1.0 nM) 5[prime]-32P-RPS8. The reactions were heated to 95°C for 1 min and then cooled to 25°C for 15 min. The reactions were initiated by adding MgCl2 to a final concentration of 3, 10, 33, 90 or 500 mM. A 1 µl aliquot from each reaction was immediately quenched in 10 µl of stop-buffer (80% formamide, 100 mM EDTA, 0.02% xylene cyanol and bromophenol blue dyes). The remaining reaction was then drawn up into 5 µl capillary tubes in 1 µl aliquots and sealed at both ends. The capillaries were placed in water and incubated at 25°C. At appropriate time intervals over a period of 3 days, capillaries were opened and the contents quenched in 10 µl of stop-buffer. The capillaries ensured that no loss of reaction volume occurred as a result of evaporation during the long incubation. The quenched aliquots were analyzed on a 20% polyacrylamide gel and the amounts of cleaved and uncleaved substrate determined by using a Molecular Dynamics radioanalytical scanner. A total of 3-5% of the substrate was cleaved in the first 3 h, reflecting contaminating substrate molecules with either oxygen or SP-phosphorothioate linkages. From 3 h to 3 days, 13-18% additional substrate was cleaved. The data for the slower cleaving fraction of substrate was fit to a linear equation (Kaleidagraph) to determine the rate of cleavage. No detectable cleavage at other phosphate linkages occurred during the 72 h incubation. Reaction rates are the averages of two determinations which varied by <3-fold.

RESULTS

The kinetically well-characterized hammerhead 8 (HH8) was used to quantitate the effects of replacing the cleavage site phosphate of the hammerhead ribozyme with an RP- or SP-phosphorothioate linkage (Fig. 1). HH8 is an excellent system in which to perform such experiments. The short substrate simplifies the separation of RP- and SP-phosphorothioate isomers. Native gel analyses do not reveal stable alternate conformations of HH8 which could potentially interfere with kinetic analysis (22). Its reaction pathway is well characterized and it has a kcat of 1/min at pH 7.5, 10 mM MgCl2 and 25°C, which reflects the chemical step (k2) (22). Under these conditions, the binding affinity of substrate to ribozyme is 50 nM (16) and thus, saturation is easily achieved. HH8 has been used to study how modifications of residue 17 affect the kinetics and thermodynamics of the reaction (24). For the reactions reported here, single turnover conditions using a saturating concentration of ribozyme (0.5 or 1.5 µM) with <1 nM substrate were used, since the available amounts of purified RP and SP thio-modified substrates were limiting.

Values of k2 were initially determined by using 32P-labeled, unmodified (S8) or thio-modified (SPS8 or RPS8) substrates at saturating ribozyme concentrations (Table 1). The value of k2 = 0.18/min for S8 at pH 7.0 agrees well with k2 = 1/min at pH 7.5 reported by Dahm et al. (25). The k2 of 0.17/min for SPS8, essentially the same as S8. In contrast, RPS8 has a k2 of only 1 × 10-5/min, which is ~18 000-fold slower than S8 and only slightly faster than 10-6/min obtained as the background rate of cleavage of RNA in a similar buffer (26,27). Since the values of k2 for all three substrates are essentially the same at 0.5 and 1.5 µM ribozyme, all three hammerheads are at saturation.

Table 1. Cleavage rates of HH8 assembled with unmodified, SPS8 or RPS8 thio-modified substrates as a function of [MgCl2]
MgCl2 (mM) k2a (/min) for substrates Thio effectb
Unmodified S8 SPS8 RPS8
3 0.12 0.06 1.5 × 10-6 80 000
10 0.18 0.17 1.0 × 10-5 18 000
33 0.69 0.40 7.0 × 10-5 10 000
90 2.4 2.5 1.4 × 10-4 16 000
500 3.0 - 1.2 × 10-3 2000
aCleavage rates of HH8 with 1.5 µM ribozyme, trace (<1.0 nM) 5[prime] end labeled substrate, 50 mM PIPES (pH 7.0) and varying amounts of MgCl2.
bThe thio effect is the ratio of the cleavage rates of the unmodified and RPS8 substrates.

In order to isolate the effect at the cleavage site from more general effects of Mn2+ on hammerhead structure, the rescue experiment should be performed in the presence of a high concentration of Mg2+ ions so that a majority of the metal binding sites are filled with Mg2+. Since some reports suggested that the RP isomer can cleave at high Mg2+ concentrations (3,4), the cleavage rates of RPS8 and SPS8 with S8 were first compared at several concentrations of Mg2+. The results of these experiments are shown in Table 1. The cleavage rate for S8 increases with the Mg2+ concentration in a manner similar to that observed previously (25). The cleavage behavior of the SP substrate is essentially the same as S8. In contrast, the RP S8 reaction remains slow at all Mg2+ concentrations. Even at 0.5 M Mg2+, the cleavage rate of RPS8 is 2000-fold slower than S8.

Since any concentration of Mg2+ can be used for the rescue experiment, we chose 50 mM, which is near saturation for HH8 (25). At this concentration, it is less likely that the rescue metals will compete for other binding sites for metals on the hammerhead which could perturb the reaction in a manner unrelated to the thio-modification. Mn2+, Co2+ and Cd2+ were tested as rescue metals. All are known to prefer sulfur over oxygen ligands relative to Mg2+, but each possess different coordination properties and could potentially fit into the cleavage site differently (14). Lower concentrations of thiophilic metals were used so that they would not compete with Mg2+ at other sites in the hammerhead and not contribute significantly to the total concentration of hydroxy cation in the reaction. At these concentrations, each of the metal ions is completely soluble at [le]pH 7.0 (18). The temperature of the reaction was also lowered to 10°C in order to slow the cleavage rate so that it could be measured accurately. The results of the initial rescue experiments are shown in Table 2. The addition of only 200 µM Mn2+ increases the rate of RPS8 cleavage by >500-fold, which agrees with the trend observed in previous experiments (3,4,12). In contrast, the oxygen substrate only increases by ~5-fold when 200 µM Mn2+ is added. Thus, it is clear that low concentrations of Mn2+ increase the rate of RPS8 substrate cleavage much more than S8 cleavage. As the concentration of Mn2+ is further increased, the cleavage rates of S8 and RPS8 both slowly increase, suggesting that Mn2+ may participate at an additional site, as well. The data for adding various Co2+ concentrations are virtually identical to the Mn2+ data; however, the Cd2+ data are somewhat different. At 200 µM Cd2+, the cleavage rate for S8 is not affected while the rate of RPS8 is slightly higher than for S8. At Cd2+ concentrations >200 µM, the cleavage rates of both S8 and RPS8 increase slowly. Therefore, low concentrations of all three thiophilic metal ions differentially stimulate the rate of RPS8 with respect to S8, even in the presence of a large excess of Mg2+. Of the three thiophilic metal ions, Cd2+ is the most effective at rescuing the cleavage rate of RPS8, which correlates well with its greater affinity for sulfur, compared with Mn2+ and Co2+ (14).

In order to more carefully investigate Cd2+ rescue, a more extensive titration was performed, using Cd2+ concentrations ranging from 2.5 to 600 µM. The reaction conditions were identical except that 25°C was used, since all the rates can be accurately measured at this temperature. As shown in Figure 2A rescue by Cd2+ is even more clearly demonstrated. When the concentration of Cd2+ is <200 µM, the rate of the S8 substrate does not change significantly. In contrast, the cleavage rate of RPS8 increases with increasing Cd2 with a Kapp for Cd2+ of ~50 µM. Thus, at 66 µM Cd2+, the cleavage rate of RpS8 is identical to that of S8 with (or without) the same concentration of Cd2+. Thus, one can conclude that Cd2+ promotes full rescue of the phosphorothioate substrate.

Table 2. Cleavage rate (k2) for S8 and RPS8 at high concentrations of thiophilic metal ions
  k2/min
[MnCl2] mM S8 RPS8
0.0 0.2 <3 × 10-4
0.2 1.1 0.1
0.6 1.4 0.1
2.0 2.1 0.2
6.6 2.8 0.6
[CoCl2] mM S8 RPS8
0.0 0.2 <3 × 10-4
0.2 1.2 0.1
0.7 1.5 0.3
2.0 (-) 0.5
6.6 2.9 (-)
[CdCl2] mM S8 RPS8
0.0 0.2 <3 × 10-4
0.2 0.2 0.5
0.7 0.4 1.0
2.0 0.4 1.5
6.6 0.9 1.2
Cleavage rates (k2) were determined at 1.5 µM ribozyme, trace 5[prime] end labeled substrate, in 50 mM PIPES (pH 7.0), 50 mM MgCl2, and the indicated concentrations of CdCl2, CoCl2 and MnCl2 at 10°C. Cleavage of the RPS8 substrate was not carried out for as long as in Table 1.

A similar Cd2+ titration experiment was performed with 100 mM Ca2+ instead of 50 mM Mg2+ (Fig. 2B). Ca2+ supports hammerhead cleavage in a manner similar to Mg2+, but the rate is 15-fold slower at a given concentration. The slower rate has been attributed to the 15-fold lower concentration of [Ca(OH)]+ in the reaction (25), but could be due to a slightly altered structure of the hammerhead in Ca2+ or to the binding of Ca2+ instead of Mg2+ to the 2[prime] oxygen (28) or another oxygen in the transition state. In 100 mM CaCl2, the cleavage rate of S8 is 0.06/min and RPS8 is 8.5 × 10-5/min giving a 700-fold thio effect. The general features of the rescue experiment are quite similar to when Mg2+ was used. Complete rescue of the RpS8 substrate is achieved between 44 and 66 µM Cd2+. However, at these concentrations of Cd2+, the cleavage rate of the S8 substrate is also slightly enhanced. Either Cd2+ is slightly more effective at competing with Ca2+ than with Mg2+ or the slower absolute rate in Ca2+ makes the reactions more sensitive to the added Cd2+.


Figure 2. The effect of CdCl2 concentration on the cleavage rate of HH8 in 50 mM PIPES (pH 7.0) and either 50 mM MgCl2 (A) or 100 mM CaCl2 (B) at 25°C with the oxygen substrate ([open circle]) and the RP-phosphorothioate substrate ([closed square]). The lines are drawn to fit the data points.

DISCUSSION

The thio-modification of the cleavage site pro-RP oxygen reduces the cleavage rate by [ge]1000-fold under various reaction conditions. Considering the total catalytic potential of the hammerhead, the effect of the thio-modified substrate is very large. At 10 mM Mg2+, the hammerhead enhances the cleavage rate ~1 000 000-fold over the non-catalyzed rate, whereas the phosphorothioate hammerhead enhances the cleavage rate by ~10-fold. Thus, the RP-phosphorothioate substitution at the cleavage site is one of a growing number of structurally moderate mutations that cause very large transition state effects in the hammerhead. For instance, an RP-phosphorothioate at position 9 reduces k2 1000-fold, and replacement of G5 with an adenosine or abasic residue causes a 100 000-fold effect (29,30). It should be noted, however, that not all structurally moderate mutations give large transition state effects in the hammerhead. For example, in a recent analysis of functional group mutations on nucleotide C17, most mutations produced much smaller transition state effects (24).

Although qualitatively in agreement with previous studies using different hammerheads, our data for HH8 indicates that an RP-thiophosphate at the cleavage site is much more deleterious than was previously indicated. Six other studies report reductions in cleavage rate varying from 25-fold to 1000-fold (Table 3). The discrepancies among the studies exist both in the reported rates for the RP-phosphorothioate substrates and in the rates of the unmodified substrates. It is unlikely that these discrepancies reflect an intrinsic difference of the RP isomer on the chemical step for the different hammerhead sequences. In recent reports, the same hammerhead mutation was examined in two or more kinetically well-characterized systems, and a very similar effect was observed (24,31). Instead, the variation among the reported thio effects probably derives from the substantial experimental difficulties in obtaining reliable kinetic data for many hammerhead ribozymes (32). One such difficulty is that some of the studies used hammerheads that formed alternate conformations, making measurement of the chemical step more difficult, even for the unmodified control hammerhead. For example, in the previous measurements of the cleavage-site thio effect from this laboratory (3), the hammerhead used contained alternate conformations in the ribozyme, and perhaps in the substrate strand, resulting in an unusually high Km (2.3 µM) (16). Thus, the reported rates were obtained under subsaturating conditions that partially reflect rate limiting association of ribozyme and substrate. Several other of the hammerheads in Table 3 also form alternate conformers, as is discussed in greater detail elsewhere (32). However, alternate conformations would not generally be expected to greatly alter the thio effect, unless the phosphorothioate substitution changed the proportion of alternate conformers.

Table 3. Comparison of the cleavage site thio effects in various hammerheads
Hammerhead kobserved/min-1a substrates Thio effectb Reference
Oxygen RP SP
HH8 0.18 10-5 0.17 18 000 This work
HH10 0.15 0.003 (n.d.) 50 (3)
R37 0.5c 0.015d 0.25 33 (4)
CL-2,4 0.25 0.0045 0.70 55 (6)
R32 0.086 0.00016 0.003 540 (20)
HH3 0.93 0.00093 0.85 1000 (12)
R34 0.25 0.01 0.12 25 (37)
akobserved refers to the rates reported in the various works for an unmodified hammerhead substrate and substrates containing either RP or SP thiophosphate at the cleavage site. The reported values do not necessarily reflect the chemical step in the hammerhead reaction.
bThe thio effect is the ratio of kobserved for the oxygen and RP-phosphorothioate substrates.
cMultiple turnover reactions containing a 1:30 ratio of ribozyme to substrate gave a t1/2 of 32/min, from which an estimated cleavage rate of 0.5/min was calculated.
dCalculated from 12% substrate cleaved in 240 min.
(n.d.) indicates that the value was not determined.

A second experimental difficulty in obtaining a reliable estimate of the thio effect at the cleavage site, derives from the inevitable presence of a small amount of contaminating SP-phosphorothioate or phosphate linkage in the Rp substrate oligonucleotide that either originates during purification or as a result of desulfurization. Thus, when measuring the cleavage rate of the Rp substrate, it is critical not to use data from the early time points which will primarily reflect cleavage of the faster cleaving impurities. This problem is compounded if the reactions are performed using substrate excess (4). In such a case, some of the impurities will not be able to bind the ribozyme and cleave until a previously bound Rp substrate cleaves or dissociates. Thus, even the faster cleaving impurities will cleave throughout the reaction, thereby overestimating the rate of cleavage of the phosphorothioate substrate. A final difficulty in obtaining a reliable measure of thio effects is that the buffer used in the reaction must not contain even trace amounts of thiophilic metals. As shown above, cleavage of the RP substrate can be observed even when a metal-impurity is present at 2000-fold lower concentrations (e.g. 2.5 µM Cd2+ in 50 mM Mg2+). In our experience, many sources of Mg2+ salts have sufficient impurities to stimulate the cleavage of the RP-phosphorothioate substrate especially when used at higher concentrations. Presumably, one or more of the above problems have led to an overestimate of the RP isomer cleavage rate in all of the previous studies. However, additional experiments would need to be done with each of the systems previously reported in order to be certain.

The very large reduction in the cleavage rate was obtained under conditions designed to avoid the problems described above. HH8 appears to be free of alternate conformations (16) and substrate dissociation is very rapid. Thus, by performing a cleavage reaction over the course of several days and only using the later time points, cleavage rate of the RP substrate can be accurately determined. It is probably best to consider these data as an upper limit of the cleavage rate for the RP isomer, since some desulfurization to oxygen probably occurs during the course of the long incubation. The rate of desulfurization for a dinucleotide phosphorothioate is 1 × 10-6/min (7), or close to the background rate of hydrolysis of RNA. A ribozyme excess experiment with a hammerhead that cannot exchange its substrate, such as hammerhead 16 (29), would be a valuable system in which to confirm this large cleavage-site thio effect.

Unlike most previous rescue experiments, the experiments here were performed in the presence of a higher concentration of a `folding' metal ion (either 50 mM MgCl2 or 100 mM CaCl2). This mixed metal ion approach has been used successfully in rescue experiments in the group I intron (10,33,34). It has several advantages over protocols which completely change the identity of the metal ion. The high concentration of the Mg2+ or Ca2+ ensures that the hammerhead is fully folded. Likewise, it ensures that most metal ion binding sites in both the thio-modified and unmodified hammerheads with Mg2+ are partially or fully saturated. As a result, the added thiophilic metal ions can only minimally interact with sites other than the one bearing the sulfur ligand. In addition, the low concentration of the thiophilic 'rescue' metal ion used in these experiments minimizes change in the total concentration of [M(OH)+], that is believed to promote cleavage (25). The only disadvantage of using the high concentration of Mg2+ or Ca2+ is that they can potentially compete with the rescue metal ion, and thereby increase the amount needed for full restoration of activity.

All three thiophilic metal ions tested (Mn2+, Co2+ and Cd2+) promote clear rescue of the hammerhead containing an RP-thiophosphate at the cleavage site. In each case, the cleavage rate increased from <10-4/min to a value that approached the oxygen control, when 200 µM metal ion was added. At 200 µM, there were small differences in the cleavage rate of the oxygen control among the three metal ions. Mn2+ and Co2+ increased the cleavage rate, whereas Cd2+ did not. This result presumably reflects the different ability of these metal ions to either bind another site on the hammerhead, or to compete with Mg2+ for the pro-RP oxygen at the cleavage site. More careful titration with Cd2+ in both Mg2+ and Ca2+ reinforces this conclusion. Cd2+ is capable of fully restoring cleavage activity for the RP substrate at concentrations as low as 66 µM, and without affecting the activity of the unmodified hammerhead. The very large stimulation of the cleavage rate in the presence of Cd2+ may simply reflect the occupation of thiophilic metal in the transition state. If so, then the corresponding binding constant for Cd2+ in the transition state is much tighter. Therefore, a more favorable transition state binding of the cleavage-site metal ion helps promote catalysis. The much tighter binding of Cd2+ in the transition state suggests that the metal ion makes interactions in the transition state that are not made in the ground state.

The precise coordination properties of this metal ion at the cleavage site of the hammerhead are not yet fully defined. In the freeze-trapped RNA hammerhead (15), a Mg2+ ion was observed within 2.2-2.4 Å of the pro-RP oxygen, which is consistent with direct coordination. No other atoms within the RNA are close enough to imply direct coordination to the Mg2+, suggesting that the remaining coordination sites are occupied by water molecules. However, the resolution of the structure is sufficiently low that additional ligands to the Mg2+ may be revealed upon further refinement. In addition, it is possible that the structure in the transition state is significantly different than in the crystal structure (29), and that additional ligands to the Mg2+ are formed in the transition state.

In many respects, our analysis of Cd2+ rescue of the phosphorothioate at the cleavage site reported here, closely resembles the recent analysis of Cd2+ rescue at phosphate 9 in the hammerhead (29). In both cases, the cleavage rate was reduced >1000-fold by the substitution of a sulfur atom. This decrease in the cleavage rate was fully rescued by Cd2+ with a Kapp in the range of 25-50 µM in the presence of relatively high Mg2+ concentrations. The presence of metal ion at phosphate 9 is also supported by X-ray crystallographic data (35,36), but the interaction is quite different, with coordination to the pro-RP oxygen and also the N7 of G10.1. While the binding of a metal ion to the cleavage site phosphate is consistent with numerous mechanistic proposals, a similar role for phosphate 9 is much less clear, considering that it is very far from the cleavage site. A large scale conformational change has been proposed that places the metal at P9 closer to the cleavage site (29).

The conclusions presented in this paper agree with a recent report examining the interaction of the very large and very thiophilic divalent ion Hg2+ with a hammerhead containing an RP-phosphorothioate at the cleavage site (37). Full rescue of the cleavage rate is observed with very low concentrations of Hg2+. Evidence for direct coordination of the Hg2+ with the sulfur is suggested by a UV absorption change that may be a charge-transfer band.

The conclusions presented in this paper disagree with those of Zhou et al. (20) who argued that all the published Mn2+ rescue data could be explained in terms of the higher concentration of metal hydroxides present in the rescue reactions due to the lower pKa of Mn2+-bound water molecules. This argument implies that Mn2+ binding to the phosphorothioate at the cleavage site does not occur. Indeed, experiments where the cleavage rate of the phosphorothioate hammerhead is measured solely in the presence of Mn2+ ion are difficult to interpret, because of the requirement for divalent ions to promote hammerhead folding. However, by using mixed metal ion protocol, we show that complete rescue can be achieved without significantly changing the total metal hydroxide concentration. For example, in the presence of 50 mM Mg2+ ion, the addition of 66 µM Cd2+ ion fully rescued the cleavage rate, while only increasing the total metal hydroxide concentration from 1.8 to 2.5 µM. As expected, no detectable change in the cleavage rate of the unmodified hammerhead was observed. Thus, the earlier conclusion that a metal ion must be bound to the RP phosphorothioate for hammerhead cleavage to occur remains valid. It is, however, important to point out that the presence of a metal ion coordinated to the pro-R oxygen in the unmodified hammerhead is only inferred. It is possible that the presence of the sulfur `recruits' the metal ion to the site and the reaction proceeds with a different mechanism than when oxygen is present. However, especially in light of the crystallographic evidence, the earlier suggestion that a metal ion is bound to the pro-RP oxygen remains reasonable and should be part of any mechanistic model for hammerhead catalysis.

The role of this metal ion in the catalytic mechanism remains unexplained. It may act as a Lewis acid to increase the susceptibility of the phosphate to nucleophilic attack by the 2[prime] oxygen. It may also help position the phosphate in its transition state structure in the way similar to that proposed for several protein enzymes (38). Additional rescue experiments with hammerheads containing sulfurs at other positions near the cleavage site phosphate should help to clarify the location and role of metal ions in the transition state structure of the hammerhead.

ACKNOWLEDGEMENT

This work was supported by grant GM 36944.

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*To whom correspondence should be addressed. Tel: +1 303 492 6929; Fax: +1 303 492 3586; Email: olke.uhlenbeck@colorado.edu

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