Prior studies on the hepatitis delta virus (HDV) ribozyme suggested a mechanistic role for the proR non-bridging oxygen on the scissile phosphate, but were unable to establish either a thio effect or a metal ion interaction. A recent crystal structure of the pre-cleaved ribozyme suggests that the proR oxygen acts as a direct ligand for a catalytic Mg2+ ion. This study probes the function of the non-bridging oxygens, utilizing phosphorothioate substitutions and metal ion rescue experiments. The Rp substrate exhibited a biphasic kinetic profile, with the fast- and slow-reacting phases displaying thio effects of ~5 and ~800, respectively, each of which was rescued with Cd2+. Experiments in the absence of divalent ions support an additional role for the proR oxygen in positioning the catalytic residue C75. Our findings support a dual role for the scissile phosphate of the HDV ribozyme in binding a metal ion and positioning a catalytic base

Importance of the scissile phosphate in orienting the catalytic metal ion and nucleobase C75 in the HDV ribozyme

245th National Meeting (2013)

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