Studies of the Catalytic-Metal-Binding Site in the Hammerhead Ribozyme Using the Phosphorothioate Approach
The phosphorothioate approach has been used widely in the literature in combination with activity assays to establish an interaction of metal ions with phosphate oxygens in the ribozyme structure. An enhancement in cleavage rate upon addition of a softer metal ion than Mg2+, such as Mn2+ is taken as evidence for coordination of the metal ion to the phosphorothioate sulfur and by analogy, an interaction of Mg 2+ with oxygen in the native ribozyme. UV-vis, EXAFS and 31 P NMR evidence for the interaction of Hg2+ with the phosphorothioate sulfur in the hammerhead ribozyme is presented here along with evidence for the unexpected discovery that soft metal ions like Hg 2+ and Mn2+ are capable of catalyzing desulfurization of the phosphorothioate at rates similar to that of ribozyme-catalyzed cleavage.
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign