Importance of magnesium ions in the mechanism of catalysis by a hammerhead ribozyme: strictly linear relationship between the ribozyme activity and the concentration of magnesium ions

ARTICLE

Auteur(s) : Atsushi Inoue^{1, 2, #}, Yasuomi Takagi^{1, 3, #}, Kazunari Taira^{1, 2,} *

¹ Gene Function Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Science City, Ibaraki 305-8562, Japan. 
² Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan. 
³ IGENE Therapeutics, Inc., C/O National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Science City, Ibaraki 305-8562, Japan. 
^# These two authors contributed equally to this work.

Introduction

Naturally existing catalytic RNAs include hammerhead, hairpin, hepatitis delta virus (HDV) and Varkud Satellite (VS) ribozymes; group I and II introns; and the RNA subunit of RNase P [1-7]. In addition, recent structural and chemical analyses strongly suggest that the ribosomal RNA is a ribozyme [8-11] and the possibility that the RNA component of the spliceosome might also be a ribozyme [12]. Since the earliest research on ribozymes, it was assumed that all ribozymes were metalloenzymes that require divalent metal ions, especially Mg²⁺ ions, for catalysis and that all must operate by a basically similar mechanism. However, extensive efforts after the discovery of ribozymes have revealed that the activity of hairpin ribozymes is independent of divalent metal ions [7, 13-18]. Thus, the various types of ribozyme appear to exploit different cleavage mechanisms, which depend upon the architecture of the individual ribozyme.

Earlier experiments with hammerhead ribozymes in vitro demonstrated that divalent metal cations are required for their activities [19-31]. These observations were consistent with the view that hammerhead ribozymes are metalloenzymes that require divalent cations for catalysis, in agreement with the earlier notion that all ribozymes might operate by a basically similar mechanism. In the case of hammerhead ribozymes (figure 1A), a large body of evidence indicates that the P9/G_10.1 site binds a metal ion with high affinity [30-42], with other metal-ion-binding sites being located around the G₅ nucleobase and A₁₃ phosphate near the site of cleavage [43, 44]. However, it was recently demonstrated that hammerhead ribozymes are active in the presence of very high concentrations of monovalent cations [45-47]. These observations raise the possibility that the hammerhead ribozyme should not be classified as a metalloenzyme and the possibility that a nucleobase of the ribozyme might act as a catalyst.

We recently observed an unusual phenomenon when we analyzed the activity of a hammerhead ribozyme as a function of the concentration of Na⁺ ions on a background of a low concentration of either Mn²⁺ or Mg²⁺ ions [48]. At lower concentrations of Na⁺ ions, Na⁺ ions had an inhibitory effect on ribozyme activity, whereas at higher concentrations, Na⁺ ions had a rescue effect. We proposed that these observations could be explained if we accept the existence of two kinds of metal-binding sites which have different affinities. Our data also supported the «two-phase folding theory» (figure 1B) [49-52], in which divalent metal ions in the ribozyme-substrate complex have lower and higher affinities, as proposed by Lilley's group on the basis of their observations of ribozyme complexes in the ground state.

In the present study, in addition to the analysis of solvent isotope effects, we carried out ribozyme reactions under single turnover conditions in the presence of high concentrations of Mg²⁺ ions, in contrast to the previously used ribozyme reactions on a background of a low concentration of either Mn²⁺ or Mg²⁺ ions [48]. Our analysis demonstrates that a catalytic magnesium ion with a very low affinity (K_d > 800 mM) exists and/or the predominant inactive complex converts to a minor active complex before the cleavage reaction.

Materials and Methods

Preparation of the hammerhead ribozyme and substrate

The ribozyme (R32) and its substrate (S11) were chemically synthesized on a DNA/RNA synthesizer using phosphoramidic chemistry with 2'-tert butyldimethylsilyl (TBDMS) protection. Chemically synthesized oligonucleotides were deprotected in 28% ammonia/ethanol (3:1) solution at 55°C for 8 hours, evaporated to dryness, redissolved in 1 ml of 1 M tetrabutylammonium fluoride at room temperature for 12 hours, and after addition of 1 ml of water, desalted on a gel-filtration column. Fully deprotected oligonucleotides were purified by gel electrophoresis in 20% polyacrylamide containing 7 M urea, and corresponding bands were excised and extracted from the gel with water. The oligonucleotides were recovered by ethanol precipitation, and then desalted using gel filtration by HPLC with ultrapure water. All of the RNA oligomers were quantified by UV absorbance at 260 nm.

The S11 was labeled with [γ-³²P] ATP by T4 polynucleotide kinase and purified by a 20% polyacrylamide gel containing 7 M urea and then purified by standard procedure as described above with desalting using gel filtration column (NAP^TM-10 column, Amersham Pharmacia Biotech, Sweden).

Measurements for the ribozyme reaction

All R32-catalyzed reactions were performed under single-turnover conditions. For the measurements of the apparent kinetic deuterium solvent isotope effect, k_obs^H2O/k_obs^D2O, we chose following conditions: 50 mM Bis-Tris propane at 25°C, 4 M NL₄Cl, pL 8 (L = H or D); 2 M LiCl, pL 7.5; 25 mM MgCl₂, pL 7. For the Mg²⁺ dependency experiment, the reaction mixture contained 25 mM Bis-Tris at pH 6.0 and 25°C.

Reactions were initiated by addition of the substrate to a mixture of metal-ion-containing buffer and the ribozyme, and aliquots were removed from the reaction mixture at appropriate intervals. These aliquots were then mixed with three times the volume of a solution that contained 100 mM MES (pH 6), 100 mM EDTA, 7 M urea, xylene cyanol (0.1%) and bromophenol blue (0.1%). Uncleaved substrate and 5'-cleaved products were separated on a 20% polyacrylamide gel containing 7 M urea and the extent of each cleavage reaction was quantified using an Image-analyzer (Storm 830; Molecular Dynamics, Sunnyvale, CA). For each reaction, an observed rate was obtained by non-linear least-squares fitting the reaction time courses using pseudo-first equation.

Results and Discussion

Our analysis, based on the kinetic solvent isotope effects (figure 2A), demonstrates that proton transfer occurs in reactions catalyzed by R32 ribozyme in the presence of high concentrations (4 M) of monovalent NH₄⁺ ions without metal ions [53], whereas no such proton transfer occurs in reactions catalyzed by R32 in the presence of divalent metal ions [54, 55]. This is based on the fact that the intrinsic isotope effect for NH₄⁺- mediated R32-catalyzed reactions is two, whereas the corresponding value for Mg²⁺- mediated and Li⁺-mediated reactions is one (figure 2A). On the basis of the idea that cleavage of the P-O bond at the 5'-site is a rate-limiting step [7, 20, 54], these results can best be explained by the mechanism shown in (figure 2B). In the case of NH₄⁺-mediated R32-catalyzed reactions, an NH₄⁺ ion neutralizes the developing negative charge in the transition state by transferring a proton to the leaving 5'-oxygen. By contrast, in the case of the Mg²⁺-mediated or high concentrations of Li⁺-mediated R32-catalyzed reactions, a respective metal ion neutralizes the developing negative charge in the transition state by coordinating directly with the leaving 5'-oxygen. These kinds of multi channels have also been reported in the case of HDV genomic ribozyme-catalyzed reactions [56].

Current research is reshaping basic theories about the roles of metal ions in reactions catalyzed by hammerhead ribozymes, and such ribozymes are no longer being viewed as true metalloenzymes [45-47]. The activity of a hammerhead ribozyme in the presence of monovalent ions has been used to argue against the hypothesis that metal ions could induce the deprotonation of 2'-OH or stabilize the leaving group directly or indirectly. The activity of a hammerhead ribozyme in the presence of Co(NH₃)₆³⁺ also showed that inner-sphere coordination is not necessary [46]. Despite the variety in the properties of divalent metal ions, monovalent metal ions, exchange-inert metal ions, and even ammonium ions, all have positive charge in common. An appropriate ratio of charge to the hammerhead ribozyme seems to be a condition for activity. Thus there exists a strong belief that the presence of a relatively dense positive charge, rather than of any particular metal ions, is the general fundamental requirement; and whether or not the positive charge plays a role in the chemical process is less important.

However, our recent findings suggest that there exists more than one channel for reactions catalyzed by the R32 hammerhead ribozyme [48, 53], and the role of metal ions can be assigned to a specific chemical process (figure 2B). With respect to the two different channels, it is clear that the divalent-metal-ion-catalyzed reaction is significantly more efficient than the monovalent-metal-ion-catalyzed reaction. Thus, extremely high concentrations of metal ions are required for the monovalent-metal-ion-catalyzed reactions. Therefore, it is likely that, under physiological conditions, hammerhead ribozymes use divalent ions as the catalytic cofactor and, thus, they act as true metalloenzymes in vivo.

We thus examined the reaction order of the R32-catalyzed reactions with respect to Mg²⁺ ions. Although a number of researchers have already reported such a ribozyme reactivity-Mg²⁺ concentration relationship [19, 50-52, 57-61], most of the experiments were carried out below 100 mM of Mg²⁺ ions and there is no information of the activity in an extremely high concentration of Mg²⁺ ions, such as 1 M. The information of the ribozyme activity under high concentrations of Mg²⁺ ions against the physiological concentration of Mg²⁺ ions is also useful in understanding the chemistry of ribozyme reactions as we might estimate the intrinsic cleavage rate to compare the enzymatic reaction to a non-enzymatic reaction and calculate more precise acceleration energy. As mentioned above, ribozyme reactions, R32-S11 (figure 1A), were performed under single-turnover conditions with R32 ribozyme saturating with respect to the substrate, S11, to ensure that the cleavage step can be followed kinetically, avoiding complications of the substrate association and the complex formation. The hammerhead reaction is known to be accelerated upon increase of pH with a slope of unity [7, 19, 53, 54, 58, 60]. The pH of reactions was also adjusted to 6.0 to slow down the reactions in order to measure the precise rate constant of fast reactions.

As shown in figure 3, the results demonstrate approximately first order dependence on the Mg²⁺ concentration. Importantly, the increasing rate constant at higher Mg²⁺ concentrations did not reach a plateau value even at as high as 1 M Mg²⁺ ions. The continuous linear increase in rate upon the addition of Mg²⁺ ions suggests the involvement of one Mg²⁺ ion that has a low affinity to the hammerhead ribozyme – substrate complex. Value of the estimated K_d turned out to be higher than 800 mM. At 800 mM of Mg²⁺ ions, the measured rate constant was 1.4 min ^– 1, and this was almost the limit of detection of fast cleavage reactions by hand manipulations.

Lilley's group analyzed the global structure of the hammerhead ribozyme-substrate complex in terms of ion-induced folding transitions by electrophoresis on non-denaturing gels, FRET and NMR [49-52]. They detected two sequential ion-dependent transitions (figure 1B). The first transition was the formation of domain II, resulting in coaxial stacking of helices II and III, which was induced by binding of a higher-affinity Mg²⁺ ion(s) (with a lower, submillimolar K_d) to the ribozyme-substrate complex. The second transition was the formation of the catalytic domain [the folding of domain I that consists of the sequence C₃U₄G₅A₆ (“uridine turn”) and the cleavage site C₁₇] of the ribozyme with resultant movement of stem I toward stem II, which is induced by binding of a lower-affinity Mg²⁺ ion(s) (with a higher, millimolar K_d). It is assumed that the ribozyme reaction proceeds in accordance with this scheme, with completion of the second transition by formation of domain I, and subsequent chemical cleavage of the scissile phosphodiester bond, with or without addition of a further divalent metal ion(s). Therefore, under the conditions of several hundred mM of Mg²⁺ ions, the formation of domain II and domain I would be completed. Taken these structural information into accounts, it seems reasonable to suggest that the very low affinity Mg²⁺ ion that we observed here might be involved in other steps besides the formation of domains I and II. The step might be a further conformational change or the binding of a catalytic species into the ribozyme complex.

Conclusion

In conclusion, our analysis demonstrates that a catalytic magnesium ion with a very low affinity (K_d > 800 mM) exists and/or the predominant inactive complex must be converted to a minor active complex before the cleavage reaction. Whichever the case, under the conditions of several mM concentrations of Mg²⁺ ions, more than 99% of the whole ribozyme – substrate complexes appear to exist in their inactive form.

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