Unconventional origin of metal ion rescue in the hammerhead ribozyme reaction: Mn2+-assisted redox conversion of 2 '-mercaptocytidine to cytidine

A specific oxygen atom in RNA is identified as a ligand for a metal ion whe n sulfur substitution of that atom shifts the metal ion specificity of the RNA-mediated process to a more thiophilic metal. Extensive discussion and d ebate have centered around whether a metal ion activates the 2'-oxygen nucl eophile during the phosphotransesterification reaction catalyzed by the ham merhead ribozyme (HH). To test this possibility, we probed the metal ion sp ecificity of HH reactions using a substrate that contained 2'-mercaptocytid ine at the cleavage site. This substrate is generated in situ from a. disul fide-protected precursor by treatment with tris(carboxyethyl)phosphine (TCE P). In HH reactions with this substrate, phosphotransesterification does no t 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 interactio n between the metal ion and the nucleophile. However, further analysis reve als that this switch in metal ion specificity does not arise because Mn2+ c oordinates sulfur more readily than Mg2+ does, but because under the assay conditions, the: 2'-mercaptocytidine residue is converted to a mixture of c ytidyl-1-beta -D-arabinofuranoside and cytidyl-1-beta -D-ribofuranoside, th e natural substrate for: the ribozyme. This conversion occurs in the absenc e of HH ribozyme, requires Mn2+ (or Co2+), O-2, 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 w ith an oxygen species to produce, the cytidyl-1-beta -D-arabinofuranoside a nd ribocytidine products. To our knowledge, this chemical transformation is unprecedented in the literature and represents a new reaction for nucleic acids., If oz is excluded from the HH reactions,,the 2'-sulfur is not modif ied in the presence of Mn2+ but is still blocked in the phosphotransesterif ication reaction, both in the forward direction as the mercapto (TSH) group or in the reverse direction as part of a cyclic phosphorothiolate. Althoug h we are unable to provide evidence for metal ion activation of the nucleop hile in the HH ribozyme reaction, this work establishes the groundwork for further use of 2'-mercaptonucleotides in biochemical analyses.