title: Involvement of a specific metal ion in the transition of the hammerhead ribozyme to its catalytic conformation.
abstract: Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by the pro-Rp oxygen of phosphate 9 and by N7 of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The Rp-, but not Sp-, phosphorothioate reduces the cleavage rate by 10(3)-fold, and the rate can be fully restored by addition of low concentrations of Cd2+, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location approximately 20 A from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd2+ binds with a Kd of 25 microM in the ground state and a Kd of 2.5 nM in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.
authors.affiliation: Department of Biochemistry, Stanford University, Stanford, California 94305-5307, USA.
authors.forename: A
authors.initials: A
authors.lastname: Peracchi
authors.forename: L
authors.initials: L
authors.lastname: Beigelman
authors.forename: E C
authors.initials: EC
authors.lastname: Scott
authors.forename: O C
authors.initials: OC
authors.lastname: Uhlenbeck
authors.forename: D
authors.initials: D
authors.lastname: Herschlag
mesh_terms: D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology*; D002384:Catalysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008961:Models, Structural; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D013873:Thionucleotides
chemical_list: D016337:RNA, Catalytic; D013873:Thionucleotides; D002104:Cadmium; D008274:Magnesium