title: A second catalytic metal ion in group I ribozyme.
abstract: Although only a subset of protein enzymes depend on the presence of a metal ion for their catalytic function, all naturally occurring RNA enzymes require metal ions to stabilize their structure and for catalytic competence. In the self-splicing group I intron from Tetrahymena thermophila, several divalent metals can serve structural roles, but only Mg2+ and Mn2+ promote splice-site cleavage and exon ligation. A study of a ribozyme reaction analogous to 5'-splice-site cleavage by guanosine uncovered the first metal ion with a definitive role in catalysis. Substitution of the 3'-oxygen of the leaving group with sulphur resulted in a metal-specificity switch, indicating an interaction between the leaving group and the metal ion. Here we use 3'-(thioinosylyl)-(3'-->5')-uridine, IspU, as a substrate in a reaction that emulates exon ligation. Activity requires the addition of a thiophilic metal ion (Cd2+ or Mn2+), providing evidence for stabilization of the leaving group by a metal ion in that step of splicing. Based on the principle of microscopic reversibility, this metal ion activates the nucleophilic 3'-hydroxyl of guanosine in the first step of splicing, supporting the model of a two-metal-ion active site.
authors.affiliation: Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
authors.forename: L B
authors.initials: LB
authors.lastname: Weinstein
authors.forename: B C
authors.initials: BC
authors.lastname: Jones
authors.forename: R
authors.initials: R
authors.lastname: Cosstick
authors.forename: T R
authors.initials: TR
authors.lastname: Cech
mesh_terms: D000818:Animals; D002104:Cadmium / Q000378:metabolism; D002384:Catalysis; D005091:Exons; D007438:Introns; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013455:Sulfur / Q000378:metabolism; D016808:Tetrahymena thermophila; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: C107916:3'-thioinosylyl-(3',5')uridine; D008670:Metals; D016337:RNA, Catalytic; D002104:Cadmium; D008345:Manganese; D013455:Sulfur; D014529:Uridine