Unconventional Origin of Metal Ion Rescue in the Hammerhead Ribozyme Reaction: Mn2+-Assisted Redox Conversion of 2‘-Mercaptocytidine to Cytidine

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Abstract

A specific oxygen atom in RNA is identified as a ligand for a metal ion when sulfur substitution of that atom shifts the metal ion specificity of the RNA-mediated process to a more thiophilic metal. Extensive discussion and debate have centered around whether a metal ion activates the 2‘-oxygen nucleophile during the phosphotransesterification reaction catalyzed by the hammerhead ribozyme (HH). To test this possibility, we probed the metal ion specificity of HH reactions using a substrate that contained 2‘-mercaptocytidine at the cleavage site. This substrate is generated in situ from a disulfide-protected precursor by treatment with tris(carboxyethyl)phosphine (TCEP). In HH reactions with this substrate, phosphotransesterification does not occur when Mg2+ is present as the only divalent cation but does occur in the presence of Mn2+. These results are consistent with a direct interaction between the metal ion and the nucleophile. However, further analysis reveals that this switch in metal ion specificity does not arise because Mn2+ coordinates sulfur more readily than Mg2+ does, but because under the assay conditions, the 2‘-mercaptocytidine residue is converted to a mixture of cytidyl-1-β-d-arabinofuranoside and cytidyl-1-β-d-ribofuranoside, the natural substrate for the ribozyme. This conversion occurs in the absence of HH ribozyme, requires Mn2+ (or Co2+), O2, and TCEP, and is inhibited by a free radical scavenger. The mechanism presumably involves a multistep free radical process, in which the key step is homolytic fission of the C2‘-sulfur bond induced by TCEP. The putative 2‘-carbon radical then reacts with an oxygen species to produce the cytidyl-1-β-d-arabinofuranoside and ribocytidine products. To our knowledge, this chemical transformation is unprecedented in the literature and represents a new reaction for nucleic acids. If O2 is excluded from the HH reactions, the 2‘-sulfur is not modified in the presence of Mn2+ but is still blocked in the phosphotransesterification reaction, both in the forward direction as the mercapto (−SH) group or in the reverse direction as part of a cyclic phosphorothiolate. Although we are unable to provide evidence for metal ion activation of the nucleophile in the HH ribozyme reaction, this work establishes the groundwork for further use of 2‘-mercaptonucleotides in biochemical analyses.

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