Divalent metal ions and the internal equilibrium of the hammerhead ribozyme

Citations Over TimeTop 14% of 1995 papers

Abstract

Thermodynamics of RNA cleavage/ligation were measured for a self-cleaving hammerhead ribozyme in the presence of Ca2+, Co2+, Mg2+, and Mn2+. The internal equilibrium, the ratio of cleaved to ligated RNA, decreases with increasing concentrations of each of the four divalent metal ions in a hyperbolic dependence that shows saturation. The metal ion dependence is not due to changes in ionic strength, and the value of the equilibrium constant at saturation is different for each metal ion. The concentration required to achieve half-saturation of the equilibrium is also different for each metal ion, and the order of apparent metal ion dissociation constants correlates with those measured for dissociation of the same metal ions complexed with tRNA and nucleotides. We interpret the divalent metal ion dependence of the equilibrium in terms of a thermodynamic model invoking noncooperative metal ion dissociation from the cleaved RNA. Thus, at 10 mM Mg2+, a commonly employed condition for hammerhead kinetic studies, metal ion dissociation contributes substantially to the free energy of the equilibrium and drives the hammerhead reaction toward cleaved RNA. Temperature dependencies of the equilibrium reveal that while the entropy and enthalpy changes of the equilibrium depend on the identity of the divalent metal ion, in each case a large entropic driving force overcomes an unfavorable change in enthalpy. This agrees with thermodynamics previously measured for an intermolecular hammerhead in the presence of Mg2+ [Hertel, K. J., & Uhlenbeck, O. C. (1995) Biochemistry 34, 1744-1749].

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