No 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.

The synthesis of an RNA dinucleotide (IspU) containing a 3‘-S-phosphorothiolate linkage is described. The compound is prepared from 9-(3-deoxy-3-iodo-β-d-xylofuranosyl)hypoxanthine with installation of the phosphorothiolate group via an Arbusov reaction and protection of the ribose 2‘-hydroxyl as a silyl ether. IspU is found to be a substrate for several enzymes including T4 polynucleotide kinase, snake venom phosphodiesterase, and ribonuclease T2. Base-catalyzed cleavage of the dinucleotide is accelerated (∼2000-fold) relative to that of the phosphate-linked compound IpU. Product characterization and kinetic analysis show that IspU is cleaved through the same mechanism as IpU. The observed rate acceleration is argued to reflect stabilization of the anionic transition state by the polarizable sulfur atom.