Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

The
hammerhead ribozyme is a well-studied nucleolytic ribozyme
that catalyzes the self-cleavage of the RNA phosphodiester backbone.
Despite experimental and theoretical efforts, key questions remain
about details of the mechanism with regard to the activation of the
nucleophile by the putative general base guanine (G12). Straightforward
interpretation of the measured activity–pH data implies the
pKa value of the N1 position in the G12
nucleobase is significantly shifted by the ribozyme environment. Recent
crystallographic and biochemical work has identified pH-dependent
divalent metal ion binding at the N7/O6 position of G12, leading to
the hypothesis that this binding mode could induce a pKa shift of G12 toward neutrality. We present computational
results that support this hypothesis and provide a model that unifies
the interpretation of available structural and biochemical data and
paints a detailed mechanistic picture of the general base step of
the reaction. Experimentally testable predictions are made for mutational
and rescue effects on G12, which will give further insights into the
catalytic mechanism. These results contribute to our growing knowledge
of the potential roles of divalent metal ions in RNA catalysis.