A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate. (original) (raw)
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Biochemistry, 2001
We describe a new RNA cleavage motif, found in the hammerhead ribozyme. Cleavage occurs between nucleotides G8 and A9, yielding a free 5'-hydroxyl group and a 2',3'-cyclic phosphate. This cleavage is dependent upon divalent metal ions and is the first evidence for a metalloribozyme known to show preference for Zn(2+). Cleavage is also observed in the presence of Ni(2+), Co(2+), Mn(2+), Cd(2+), and Pb(2+), while negligible cleavage was detected in the presence of the alkaline-earth metal ions Mg(2+), Ca(2+), Sr(2+), and Ba(2+). A linear relationship between the logarithm of the rate and pH was observed for the Zn(2+)-dependent cleavage, which is indicative of proton loss in the cleavage mechanism, either prior to or in the rate-determining step. We postulate that a zinc hydroxide complex, bound to the known A9/G10.1 metal ion binding site, abstracts the proton from the 2'-hydroxyl group of G8, which attacks the A9 phosphate and initiates cleavage. This hypothesis is supported by a previously reported crystal structure [Murray, J. B., Terwey, D. P., Maloney, L., Karpeisky, A., Usman, N., Beigelman, L., and Scott, W. G. (1998) Cell 92, 665-673], which shows the conformation required for RNA cleavage and proximity of the 2'-hydroxyl group to the metal ion complex.
Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the influence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.
Journal of Biological Chemistry, 1997
Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by the pro-R p oxygen of phosphate 9 and by N 7 of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The R p -, but not S p -, phosphorothioate reduces the cleavage rate by 10 3 -fold, and the rate can be fully restored by addition of low concentrations of Cd 2؉ , a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location ϳ20 Å from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd 2؉ binds with a K d of 25 M in the ground state and a K d of 2.5 nM in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.
Nucleic Acids Research, 1997
In the presence of magnesium ions, cleavage by the hammerhead ribozyme RNA at a specific residue leads to 2′3′-cyclic phosphate and 5′-OH extremities. In the cleavage reaction an activated ribose 2′-hydroxyl group attacks its attached 3′-phosphate. Molecular dynamics simulations of the crystal structure of the hammerhead ribozyme, obtained after flash-freezing of crystals under conditions where the ribozyme is active, provide evidence that a µ-bridging OHion is located between two Mg 2+ ions close to the cleavable phosphate. Constrained simulations show further that a flip from the C3′-endo to the C2′-endo conformation of the ribose at the cleavable phosphate brings the 2′-hydroxyl in proximity to both the attacked phosphorous atom and the µ-bridging OHion. Thus, the simulations lead to a detailed new insight into the mechanism of hammerhead ribozyme cleavage where a µ-hydroxo bridged magnesium cluster, located on the deep groove side, provides an OHion that is able to activate the 2′-hydroxyl nucleophile after a minor and localized conformational change in the RNA.
The role of metal cations (Mg2+) in the cleavage reaction of fully hydrated RNA enzymes is investigated via Car−Parrinello calculations. We find that the action of two metal catalysts is the most efficient way to promote, on one hand, the proton abstraction from O2‘−H that triggers the nucleophilic attack and, on the other hand, the weakening and subsequent cleavage of the P−O5‘ bond. The elimination of one of the two metal cations is shown to lead to an increase in the activation energy. Furthermore, we also find that an OH- included in the coordination shell of the Mg2+ close to O2‘ promotes the initial proton abstraction and prevents its transfer to the ribozyme in both single- and double-metal-ion pathways, consistently with the experiment. This suggests that in real ribozyme systems, the double-metal-ion reaction mechanism in the presence of an OH- anion is favored with respect to single-metal-ion mechanisms.
Crucial Roles of Two Hydrated Mg 2+ Ions in Reaction Catalysis of the Pistol Ribozyme
Angewandte Chemie International Edition, 2019
Pistol ribozymes constitute anew class of small selfcleaving RNAs.Crystal structures have been solved, providing three-dimensional snapshots along the reaction coordinate of pistol phosphodiester cleavage,c orresponding to the precatalytic state,avanadate mimic of the transition state,a nd the product. The results led to the proposed underlying chemical mechanism. Importantly,ahydrated Mg 2+ ion remains innersphere-coordinated to N7 of G33 in all three states,a nd is consistent with its likely role as acid in general acid base catalysis (d and b catalysis). Strikingly,t he new structures shed light on as econd hydrated Mg 2+ ion that approaches the scissile phosphate from its binding site in the pre-cleavage state to reacho ut for water-mediated hydrogen bonding in the cyclophosphate product. The major role of the second Mg 2+ ion appears to be the stabilization of product conformation. This study delivers amechanistic understanding of ribozyme-catalyzed backbone cleavage. Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone.T hey are widely distributed in nature and are essential for rolling-circle-based replication of satellite and pathogenic RNAs. [1-13] Comparative genomic analysis led to the discovery of novel selfcleaving ribozymes,n amed twister,t wister-sister, pistol, and hatchet. [14, 15] Forthe first three classes,the three-dimensional architectures [16-25] in pre-cleavage states were solved by X-ray crystallography,a nd very recently,a lso the first structure of ah atchet ribozyme (product) has been determined. [26] These structures represent athorough basis to explore the chemical mechanism of the site-specific transesterification reactions [6,27] that are catalyzed by these ribozymes (Figure 1a).
Journal of Chemical Theory and Computation, 2007
Results of a series of 12 ns molecular dynamics (MD) simulations of the reactant state (with and without a Mg 2+ ion), early and late transition state mimics are presented based on a recently reported crystal structure of a full-length hammerhead RNA. The simulation results support a catalytically active conformation with a Mg 2+ ion bridging the A9 and scissile phosphates. In the reactant state, the Mg 2+ spends significant time closely associated with the 2′OH of G8, but remains fairly distant from the leaving group O 5′ position. In the early TS mimic simulation, where the nucleophilic O 2′ and leaving group O 5′ are equidistant from the phosphorus, the Mg 2+ ion remains tightly coordinated to the 2′OH of G8, but is positioned closer to the O 5′ leaving group, stabilizing the accumulating charge. In the late TS mimic simulation, the coordination around the bridging Mg 2+ ion undergoes a transition whereby the coordination with the 2′OH of G8 is replace by the leaving group O 5′ that has developed significant charge. At the same time, the 2′OH of G8 forms a hydrogen bond with the leaving group O 5′ and is positioned to act as a general acid catalyst. This work represents the first reported simulations of the full-length hammerhead structure and TS mimics, and provides direct evidence for the possible role of a bridging Mg 2+ ion in catalysis that is consistent with both crystallographic and biochemical data.
Biochemistry, 1999
The hammerhead ribozyme crystal structure identified a specific metal ion binding site referred to as the P9/G10.1 site. Although this metal ion binding site is ∼20 Å away from the cleavage site, its disruption is highly deleterious for catalysis. Additional published results have suggested that the pro-R P oxygen at the cleavage site is coordinated by a metal ion in the reaction's transition state. Herein, we report a study on Cd 2+ rescue of the deleterious phosphorothioate substitution at the cleavage site. Under all conditions, the Cd 2+ concentration dependence can be accounted for by binding of a single rescuing metal ion. The affinity of the rescuing Cd 2+ is sensitive to perturbations at the P9/G10.1 site but not at the cleavage site or other sites in the conserved core. These observations led to a model in which a metal ion bound at the P9/G10.1 site in the ground state acquires an additional interaction with the cleavage site prior to and in the transition state. A titration experiment ruled out the possibility that a second tightbinding metal ion (K d Cd < 10 µM) is involved in the rescue, further supporting the single metal ion model. Additionally, weakening Cd 2+ binding at the P9/G10.1 site did not result in the biphasic binding curve predicted from other models involving two metal ions. The large stereospecific thio-effects at the P9/ G10.1 and the cleavage site suggest that there are interactions with these oxygen atoms in the normal reaction that are compromised by replacement of oxygen with sulfur. The simplest interpretation of the substantial rescue by Cd 2+ is that these atoms interact with a common metal ion in the normal reaction. Furthermore, base deletions and functional group modifications have similar energetic effects on the transition state in the Cd 2+ -rescued phosphorothioate reaction and the wild-type reaction, further supporting the model that a metal ion bridges the P9/G10.1 and the cleavage site in the normal reaction (i.e., with phosphate linkages rather than phosphorothioate linkages). These results suggest that the hammerhead undergoes a substantial conformational rearrangement to attain its catalytic conformation. Such rearrangements appear to be general features of small functional RNAs, presumably reflecting their structural limitations.
Cleavage reaction of HDV ribozymes in the presence of Mg2+ is accompanied by a conformational change
Genes To Cells, 2002
Background: Hepatitis delta virus (HDV) ribozymes cleave RNA in the presence of divalent metal ions. We have previously elucidated the solution conformation of a minimized transacting HDV ribozyme and obtained evidence by NMR study that an Mg 2+ ion binds to a site close to the cleavage site. Results: We examined two ribozyme systems: a pre-cleavage complex with a non-cleavable substrate analogue (mS8) and a post-cleavage complex with a 3′ ′ ′ ′ cleavage product (P7). Upon titration with MgCl 2 , the complex with P7 showed a profound spectral change, while that with mS8 showed broadening of the signals. Analysis of the NOESY spectra of the P7 complex at high Mg 2+ concentration revealed that a G:U pair is formed within the L3 loop, and the P1 and P4 stems are stabilized with respect to those of the pre-cleavage complex. Conclusion: The present analysis indicates that the cleavage reaction of the HDV ribozyme produces a big conformational change. Furthermore, presence of the 5′ ′ ′ ′-terminal cytidine residue prevents this conformational change and its absence stabilizes the productribozyme complex in the presence of Mg 2+. The structure of the Mg 2+-bound P7 complex is similar to the crystal structure found for a product-ribozyme complex but is different from the pre-cleavage structure.
Journal of the American Chemical Society, 2003
The hammerhead ribozyme is an RNA molecule capable of self-cleavage at a unique site within its sequence. Hydrolysis of this phosphodiester linkage has been proposed to occur via an in-line attack geometry for nucleophilic displacement by the 2′-hydroxyl on the adjoining phosphorus to generate a 2′,3′cyclic phosphate ester with elimination of the 5′-hydroxyl group, requiring a divalent metal ion under physiological conditions. The proposed SN2(P) reaction mechanism was investigated using density functional theory calculations incorporating the hybrid functional B3LYP to study this metal ion-dependent reaction with a tetraaquo magnesium (II)-bound hydroxide ion. For the Mg 2+ -catalyzed reaction, the gas-phase geometry optimized calculations predict two transition states with a kinetically insignificant, yet clearly defined, pentacoordinate intermediate. The first transition state located for the reaction is characterized by internal nucleophilic attack coupled to proton transfer. The second transition state, the rate-determining step, involves breaking of the exocyclic P-O bond where a metal-ligated water molecule assists in the departure of the leaving group. These calculations demonstrate that the reaction mechanism incorporating a single metal ion, serving as a Lewis acid, functions as a general base and can afford the necessary stabilization to the leaving group by orienting a water molecule for catalysis.