A 2ND CATALYTIC METAL-ION IN A GROUP-I RIBOZYME

Although only a subset of protein enzymes depend on the presence of a metal ion for their catalytic function, all naturally occurring RNA en zymes require metal ions to stabilize their structure and for catalyti c competence(1). In the self-splicing group I intron from Tetrahymena thermophila(2), several divalent metals can serve structural roles, bu t only Mg2+ and Mn2+ promote splice-site cleavage and exon ligation(3, 4). A study of a ribozyme reaction analogous to 5'-splice-site cleavag e by guanosine uncovered the first metal ion with a definitive role in catalysis. Substitution of the 3'-oxygen of the leaving group with su lphur resulted in a metal-specificity switch, indicating an interactio n between the leaving group and the metal ion(5). Here we use 3'-(thio inosylyl)-(3' --> 5')-uridine(6), IspU, as a substrate in a reaction t hat emulates exon ligation. Activity requires the addition of a thioph ilic metal ion (Cd2+ or Mn2+), providing evidence for stabilization of the leaving group by a metal ion in that step of splicing. Based on t he principle of microscopic reversibility, this metal ion activates th e nucleophilic 3'-hydroxyl of guanosine in the first step of splicing, supporting the model of a two-metal-ion active site(7).