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

A novel metal-binding site has been identified in the hammerhead ribozyme b y 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 prev iously identified metal-binding sites. P-31 NMR spectroscopy was used to me asure the metal-binding affinity for this site and leads to an apparent dis sociation 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 si te upon metal binding and these results are compared with previous data on metal-induced structural changes in the core of the hammerhead ribozyme, Th ese NMR data were combined with the X-ray structure of the hammerhead riboz yme (Pley HW, Flaherty KM, McKay DB. 1994, Nature 372:68-74) to model RNA l igands 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 demons trate that P-31 NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.