title: Identification and characterization of a novel high affinity metal-binding site in the hammerhead ribozyme.
abstract: A novel metal-binding site has been identified in the hammerhead ribozyme by 31P NMR. The metal-binding site is associated with the A13 phosphate in the catalytic core of the hammerhead ribozyme and is distinct from any previously identified metal-binding sites. 31P NMR spectroscopy was used to measure the metal-binding affinity for this site and leads to an apparent dissociation constant of 250-570 microM at 25 degrees C for binding of a single Mg2+ ion. The 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 A13 site. In this model, the A13 metal-binding site is structurally similar to the previously identified A(g) metal-binding site and illustrates the symmetrical nature of the tandem G x A base pairs in domain 2 of the hammerhead ribozyme. These results demonstrate that 31P NMR represents an important method for both identification and characterization of metal-binding sites in nucleic acids.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado at Boulder, 80309-0215, USA.
authors.forename: M R
authors.initials: MR
authors.lastname: Hansen
authors.forename: J P
authors.initials: JP
authors.lastname: Simorre
authors.forename: P
authors.initials: P
authors.lastname: Hanson
authors.forename: V
authors.initials: V
authors.lastname: Mokler
authors.forename: L
authors.initials: L
authors.lastname: Bellon
authors.forename: L
authors.initials: L
authors.lastname: Beigelman
authors.forename: A
authors.initials: A
authors.lastname: Pardi
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004305:Dose-Response Relationship, Drug; D007477:Ions; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010710:Phosphates / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry*
chemical_list: D007477:Ions; D010710:Phosphates; D016337:RNA, Catalytic; D008274:Magnesium