Ribozymes provide an observation window into the ability of RNA to create structural, thermodynamic, and electronic environments that guide function. We have applied multiple approaches to probe the active site of the Hammerhead ribozyme (HHRz).  The influence of metal ion substitution, in combination with thio substitutions and pH-dependent profiles, demonstrates metal coordination to the scissile phosphate in the ground state of the reaction, and metal ion dependence of at least one protonation step in the reaction. As a geometry-specific probe and novel crosslinking reagent, Pt(II)-induced crosslinking with a phosphorothioate-substituted active site has identified conformational properties for metal ions coordinated to the scissile phosphate.  Fluorescence quenching from multiple 2-AP reporters senses a single global folding transition and a distinct change in dynamics at the ribozyme active site. Taken together, these results demonstrate the synergistic influence of metal ion cofactors on local structure, dynamics, and chemistry of this ribozyme.

Analysis of a ribozyme active site: Metal-dependent kinetics, fluorescence, and crosslinking

245th National Meeting (2013)

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