Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme

A novel metal-binding site has been identified in the hammerhead ribozyme by P-31 NMR, The metal-binding site is associated with the A,, phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. P-31 NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 mu M at 25 degrees C for binding of a single Mg2+ ion. Th, NMR data also show evidence of a structural change at this site upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme, These NMR data were combined with the X-ray structure of the hammerhead ribozyme (Pley HW, Flaherty KM, McKay DB. 1994, Nature 372:68-74) to model RNA ligands involved in binding the metal at this Al, site. In this model, the A,, metal-binding site is structurally similar to the previously identified A, metal-binding site and illustrates the symmetrical nature of the tandem G.A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that P-31 NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.