Divalent metal ions tune the self-splicing reaction of the yeast mitochondrial group II intron Sc.ai5γ

Abstract

Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg²⁺ to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis–Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5′-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca²⁺, Mn²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Pb²⁺, and [Co(NH₃)₆]³⁺ on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5γ. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg²⁺. For example, the presence of only 5% Ca²⁺ relative to Mg²⁺ results in a decrease in the maximal turnover rate k _cat by 50%. Ca²⁺ thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg²⁺ in the folded state, the latter being indicative of at least one specific Ca²⁺ binding pocket interfering directly with catalysis. Similar results are obtained with Mn²⁺, Cd²⁺, and [Co(NH₃)₆]³⁺. Ni²⁺ is a much more powerful inhibitor and the presence of either Zn²⁺ or Pb²⁺ leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg²⁺ and raises the question of biological relevance at least in the case of Ca²⁺.