The hammerhead cleavage reaction in monovalent cations

Curtis, Edward A.; Bartel, David P.

doi:10.1017/s1355838201002357

Cited by 150 publications

(170 citation statements)

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“…Initially, the pH dependence of the cleavage rate constant suggested that a metal hydroxide bound to the ribozyme acts as a general base in catalysis (18). Subsequently, the catalytic activity of the hammerhead ribozyme in the presence of monovalent cations alone was found to rule out an obligatory role of divalents (19)(20)(21). Cleavage kinetics in crystals indicate that the pH dependence arises from a metal-induced rate-limiting conformational change, rather than a chemical step (22).…”

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Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

et al. 2003

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The hammerhead ribozyme is one of the best-studied small RNA enzymes, yet is mechanistically still poorly understood. We measured the Mg 2+ dependencies of folding and catalysis for two distinct hammerhead ribozymes, HHL and HHR. HHL has three long helical stems and was previously used to characterize Mg 2+ -induced folding. HHR has shorter stems and an A‚U tandem next to the cleavage site that increases activity ∼10-fold at 10 mM Mg 2+ . We find that both ribozymes cleave with fast rates (5-10 min -1 , at pH 8 and 25°C) at nonphysiologically high Mg 2+ concentrations, but with distinct Mg 2+ dissociation constants for catalysis: 90 mM for HHL and 10 mM for HHR. Using time-resolved fluorescence resonance energy transfer, we measured the stem I-stem II distance distribution as a function of Mg 2+ concentration, in the presence and absence of 100 mM Na + , at 4 and 25°C. Our data show two structural transitions. The larger transition (with Mg 2+ dissociation constants in the physiological range of ∼1 mM, below the catalytic dissociation constants) brings stems I and II close together and is hindered by Na + . The second, globally minor, rearrangement coincides with catalytic activation and is not hindered by Na + . Additionally, the more active HHR exhibits a higher flexibility than HHL under all conditions. Finally, both ribozyme-product complexes have a bimodal stem I-stem II distance distribution, suggesting a fast equilibrium between distinct conformers. We propose that the role of diffusely bound Mg 2+ is to increase the probability of formation of a properly aligned catalytic core, thus compensating for the absence of naturally occurring kissing-loop interactions.Hammerhead ribozymes are found in a number of plant pathogenic viroids and virusoids, where they are involved in self-cleavage and self-ligation of the plus and minus strands during the pathogen's genome replication (1, 2). In addition, the hammerhead ribozyme has been found to mediate self-cleavage of satellite DNA transcripts in the newt (3). Several hammerhead crystal structures have been determined (4-6), and the global structure was confirmed in solution by steady-state fluorescence resonance energy transfer (FRET) 1 (7,8), gel electrophoresis (9), and transient electric birefringence (10) (Figure 1). The ribozyme consists of three helical stems arranged in a "Y-shape", with 11 nucleotides at the junction forming the highly conserved catalytic core (11,12) (Figure 1). The catalytic core is comprised of two domains; stems I and II are arranged in an acute angle by a sharp uridine turn in the backbone trajectory of domain 1, while stems II and III stack coaxially with the formation of two G‚A and one A‚U non-WatsonCrick base pairs in domain 2. The cleavage site is located 3′ to C17, which is buttressed to domain 1 by a single hydrogen bond with C3 (Figure 1). † This work was supported by NIH Grant GM62357-01 to N.G.W., a postdoctoral fellowship from the Swiss National Funds to D.R., a DAAD scholarship to K.W., and a University of Michig...

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Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

et al. 2003

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“…Divalent metal ions can also play a structural role in ribozyme activity by stabilizing the catalytically active tertiary conformation through coordination to heteroatoms, such as oxygen atoms or nitrogen atoms present in the nucleoside bases. The finding that the hammerhead ribozyme is active in the absence of divalent metal ions at high concentrations of monovalent metal ions calls into question the role of divalent metal ions in the mechanism of substrate cleavage (5,7,8).Divalent metal ion binding sites in the hammerhead ribozyme have been found by X-ray crystallography (9-13) and by NMR spectroscopy (14). One of the most extensively studied metal ion binding sites is the A9/G10.1 site which involves coordination of the metal ion to N7 of G10.1 and the pro-R P phosphate oxygen of A9 (Figure 2).…”

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Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

2001

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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.The hammerhead ribozyme ( Figure 1A) is an RNA motif that catalyzes the cleavage of an RNA substrate via a transesterification reaction, producing a 2′,3′-cyclic phosphate and a 5′-hydroxyl terminus ( Figure 1B) (1). Divalent metal ions have been implicated directly in the hammerhead ribozyme cleavage mechanism (2-5), and the most popular mechanism has involved the abstraction of the 2′-hydroxyl proton at the cleavage site by a metal ion hydroxide (6). Divalent metal ions can also play a structural role in ribozyme activity by stabilizing the catalytically active tertiary conformation through coordination to heteroatoms, such as oxygen atoms or nitrogen atoms present in the nucleoside bases. The finding that the hammerhead ribozyme is active in the absence of divalent metal ions at high concentrations of monovalent metal ions calls into question the role of divalent metal ions in the mechanism of substrate cleavage (5,7,8).Divalent metal ion binding sites in the hammerhead ribozyme have been found by X-ray crystallography (9-13) and by NMR spectroscopy (14). One of the most extensively studied metal ion binding sites is the A9/G10.1 site which involves coordination of the metal ion to N7 of G10.1 and the pro-R P phosphate oxygen of A9 (Figure 2). The pro-R P oxygen of A9 has been implicated in hammerhead ribozyme function by replacement of the nonbridging, pro-R P oxygen at the A9 phosphate with sulfur. Substrate cleavage studies in the presence of Mg 2+ resulted in reduced † This work was supported by a grant from the National Institutes of Health (GM56947).

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“…However, further studies have shown that high concentrations of monovalent ions, including sodium (Na 1 ), ammonium (NH 41 ) and lithium (Li 1 ) not only can promote the adoption of the catalytically active fold but also can activate the ribozyme (121,122). For example, although the cleavage of the hammerhead proceeds more efficiently in divalent cations, the reaction in 4 M LiCl is just 10-fold slower than that under standard conditions of 10 mM MgCl 2 (56).…”

Section: Modulation Of Hammerhead Ribozyme Activitymentioning

confidence: 99%

Insights into functional modulation of catalytic RNA activity

et al. 2008

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SummaryRNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e., micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.

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The hammerhead cleavage reaction in monovalent cations

Abstract: 31 also supports substantial hammerhead activity. These results suggest that a metal ion does not act as a base in the reaction, and that the effects of different metal ions on hammerhead cleavage rates primarily reflect structural contributions to catalysis.

Cited by 150 publications

References 45 publications

Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

Insights into functional modulation of catalytic RNA activity

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The hammerhead cleavage reaction in monovalent cations

Abstract: 31 also supports substantial hammerhead activity. These results suggest that a metal ion does not act as a base in the reaction, and that the effects of different metal ions on hammerhead cleavage rates primarily reflect structural contributions to catalysis.

Cited by 150 publications

References 45 publications

Diffusely Bound Mg2+ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Diffusely Bound Mg2+ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme

Insights into functional modulation of catalytic RNA activity

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Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core

Diffusely Bound Mg²⁺ Ions Slightly Reorient Stems I and II of the Hammerhead Ribozyme To Increase the Probability of Formation of the Catalytic Core