Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

Inoue, Atsushi; Takagi, Yoko; Taira, Kazunari

doi:10.1093/nar/gkh753

Cited by 15 publications

(21 citation statements)

References 64 publications

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“…Minimal hRz exhibit proficient magnesiumindependent cleavage at high concentrations of monovalent ions (Murray et al 1998;Curtis and Bartel 2001), although even in 2 M Li + , the reaction is stimulated by Mg 2+ ions that appear to bind with low affinity (Inoue et al 2004). Discrete Mg 2+ binding sites have been suggested in analyses by crystallography, thiophilic metal ion rescue, EPR, and NMR (Scott et al 1996;Peracchi et al 1997;Hansen et al 1999;Hunsicker and DeRose 2000;Hoogstraten et al 2002;Wang et al 2004).…”

Section: Discussionmentioning

confidence: 99%

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Saksmerprome

Roychowdhury-Saha²,

Jayasena³

et al. 2004

RNA

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Tertiary stabilizing motifs (TSMs) between terminal loops or internal bulges facilitate folding of natural hammerhead ribozymes (hRz) under physiological conditions. However, both substrate and enzyme strands contribute nucleotides to the TSMs of trans-cleaving hRz, complicating the design of hRz that exploit TSMs to target specific mRNA. To overcome this limitation, we used SELEX to identify new, artificial TSMs that are less sensitive to sequence context. Nucleotides in loop II or in a bulge within the ribozyme strand of stem I were randomized, while the interaction partner was held constant. All nucleotides of the substrate pair with the ribozyme, minimizing their possible recruitment into the TSM, as such recruitment could constrain choice of candidate target sequences. Six cycles of selection identified cis-acting ribozymes that were active in 100 µM MgCl 2 . The selected motifs partially recapitulate TSMs found in natural hRz, suggesting that the natural motifs are close to optimal for their respective contexts. Ribozyme "RzB" showed enhanced thermal stability by retaining trans-cleavage activity at 80°C in 10 mM MgCl 2 and at 70°C in 2 mM MgCl 2 . A variant of ribozyme "RzB" with a continuously paired stem 1 rapidly lost activity as temperature was increased. The selected motifs are modular, in that they permit trans-cleavage of several substrates in submillimolar MgCl 2 , including two substrates derived from the U5 genomic region of HIV-1. The new, artificial tertiary stabilized hRz are thus nearly independent of sequence context and enable for the first time the use of highly active hRz targeting almost any mRNA at physiologically relevant magnesium concentrations.

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Section: Discussionmentioning

confidence: 99%

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Saksmerprome

Roychowdhury-Saha²,

Jayasena³

et al. 2004

RNA

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“…[74][75][76] In the single cation mechanism, the Mg 2+ preserves the integrity of the active site structure, and may serve as an epicenter in the transition state that coordinates the A9 and scissile phosphates, G8 2′OH general acid and O 5′ leaving group. The present work underscores the need for further investigation of the chemical reaction profile using combined QM/MM models.…”

Section: Mechanistic Interpretationmentioning

confidence: 99%

Role of Mg²⁺ in Hammerhead Ribozyme Catalysis from Molecular Simulation

et al. 2008

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Molecular dynamics simulations have been performed to investigate the role of Mg 2+ in the fulllength hammerhead ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/ MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg 2+ ion. The simulations suggest that Mg 2+ is profoundly involved in the hammerhead ribozyme mechanism both at structural and catalytic levels. Binding of Mg 2+ in the active site plays a key structural role in the stabilization of stem I and II and to facilitate formation of near attack conformations and interactions between the nucleophile and G12, the implicated general base catalyst. In the transition state, Mg 2+ binds in a bridging position where it stabilizes the accumulated charge of the leaving group while interacting with the 2′OH of G8, the implicated general acid catalyst. The QM/MM simulations provide support that, in the late transition state, the 2′OH of G8 can transfer a proton to the leaving group while directly coordinating the bridging Mg 2+ ion. The present study provides evidence for the role of Mg 2+ in hammerhead ribozyme catalysis. The proposed simulation model reconciles the interpretation of available experimental structural and biochemical data, and provides a starting point for more detailed investigation of the chemical reaction path with combined QM/MM methods.

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“…[32,33,34] The Mg 2+ preserves the integrity of the active site structure, and may serve as an epicenter in the transition state that coordinates the A9 and scissile phosphates, G8 2′OH general acid and O 5′ leaving group. The present work underscores the need for further investigation of the chemical reaction profile using combined QM/MM models.…”

Section: Comparison With Experimentsmentioning

confidence: 99%

Insight into the Role of Mg²⁺ in Hammerhead Ribozyme Catalysis from X-ray Crystallography and Molecular Dynamics Simulation

López

Martick

et al. 2007

J. Chem. Theory Comput.

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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.The hammerhead ribozyme [1] is an archetype system to study RNA catalysis. [2,3] A detailed understanding of the hammerhead mechanism provides insight into the inner workings of more complex cellular catalytic RNA machinery such as the ribosome, and ultimately may aid the rational design of new medical therapies [4] and biotechnology. [5,6] Despite a tremendous amount of experimental and theoretical effort, [1,2,7,8] the details of the hammerhead ribozyme mechanism have been elusive. In particular, one of the main puzzles involves the apparent inconsistency between the interpretation of thio effect experiments [9,10] and mutational data [8] with available crystallographic structural information of the minimal hammerhead sequence. [11,12,13] Results from the biochemical experiments suggest that a pH-dependent conformational change, inconsistent with crystallographic data, [11,12,13] must precede or be concomitant with the catalytic chemical step. This includes a possible metal ion bridge between the A9 and scissile phosphates that in previous crystal structures were ~20 Å apart. Moreover, the function of the 2′OH group of G8 remains unclear. A stable Mg 2+ ion bridge between the A9 and scissile phosphates is formed in the catalytically active conformationThe simulation results support a catalytic role for a Mg 2+ ion bridging the A9 and scissile phosphates. In the simulations with a bridging Mg 2+ ion, the average distance between the A9 and scissile phosphates remain within the crystallographic value of 4.3 Å, whereas in the absence of Mg 2+ this key contact between stems I and II drifts to over 7 ...

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Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

Cited by 15 publications

References 64 publications

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Role of Mg²⁺ in Hammerhead Ribozyme Catalysis from Molecular Simulation

Insight into the Role of Mg²⁺ in Hammerhead Ribozyme Catalysis from X-ray Crystallography and Molecular Dynamics Simulation

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Importance in catalysis of a magnesium ion with very low affinity for a hammerhead ribozyme

Cited by 15 publications

References 64 publications

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Artificial tertiary motifs stabilize trans-cleaving hammerhead ribozymes under conditions of submillimolar divalent ions and high temperatures

Role of Mg2+ in Hammerhead Ribozyme Catalysis from Molecular Simulation

Insight into the Role of Mg2+ in Hammerhead Ribozyme Catalysis from X-ray Crystallography and Molecular Dynamics Simulation

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Role of Mg²⁺ in Hammerhead Ribozyme Catalysis from Molecular Simulation

Insight into the Role of Mg²⁺ in Hammerhead Ribozyme Catalysis from X-ray Crystallography and Molecular Dynamics Simulation