Structural requirement for Mg²⁺ binding in the group I intron core

Divalent metal ions are required for splicing of group I introns, but their role in maintaining the structure of the active site is still under investigation. Ribonuclease and hydroxyl radical footprinting of a small group I intron from Azoarcus pre-tRNA^Ile showed that tertiary interactions between helical domains are stable in a variety of cations. Only Mg²⁺, however, induced a conformational change in the intron core that correlates with self-splicing activity. Three metal ion binding sites in the catalytic core were identified by Tb(III)-dependent cleavage. Two of these are near bound substrates in a three-dimensional model of the ribozyme. A third metal ion site is near an A minor motif in P3. In the pre-tRNA, Tb³⁺ cleavage was redirected to the 5′ and 3′ splice sites, consistent with metal-dependent activation of splice site phosphodiesters. The results show that many counterions induce global folding, but organization of the group I active site is specifically linked to Mg²⁺ binding at a few sites.