Use of phosphorothioates to identify sites of metal-ion binding in RNA

Single atom substitutions provide an exceptional opportunity to investigate RNA structure and function. Replacing a phosphoryl oxygen with a sulfur represents one of the most common and powerful single atom substitutions and can be used to determine the sites of metal-ion binding. Using functional assays of ribozyme catalysis, based on pre-steady-state kinetics, it is possible to extend this analysis to the transition state, capturing ligands for catalytic metal ions in this fleeting state. In conjunction with data determined from X-ray crystallography, this technique can provide a picture of the environment surrounding catalytic metal ions in both the ground state and the transition state at atomic resolution. Here, we describe the principles of such analysis, explain limitations of the method, and provide a practical example based on our results with the Tetrahymena group I ribozyme.