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A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

Lott, W. B., Pontius, B. W. , & von Hippel, P. H. (1998) A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate. Proceedings of the National Academy of Sciences of the United States of America (PNAS) ISSN 1091-6490, 95(2), pp. 542-547.

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Abstract

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.

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ID Code: 52752
Item Type: Journal Article
Additional Information: Lott, W B Pontius, B W von Hippel, P H GM-15792/GM/NIGMS NIH HHS/United States GM-29158/GM/NIGMS NIH HHS/United States Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. United states Proceedings of the National Academy of Sciences of the United States of America Proc Natl Acad Sci U S A. 1998 Jan 20;95(2):542-7.
Keywords: Binding Sites, Metals/ chemistry/metabolism, RNA/chemistry/ metabolism, RNA, Catalytic/chemistry/ metabolism, Substrate Specificity
ISSN: 0027-8424
Divisions: Current > Schools > School of Chemistry, Physics & Mechanical Engineering
Current > Institutes > Institute of Health and Biomedical Innovation
Current > QUT Faculties and Divisions > Science & Engineering Faculty
Copyright Owner: Copyright 1998 National Academy of Sciences.
Deposited On: 26 Jul 2012 16:10
Last Modified: 26 Jul 2012 16:10

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