Divalent metal ions tune the self-splicing reaction of the yeast mitochondrial group II intron Sc.ai5gamma.
Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg(2+) 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 Ca(2+), Mn(2+), Ni(2+), Zn(2+), Cd(2+), Pb(2+), and [Co(NH(3))(6)](3+) on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5gamma. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg(2+). For example, the presence of only 5% Ca(2+) relative to Mg(2+) results in a decrease in the maximal turnover rate k (cat) by 50%. Ca(2+) thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg(2+) in the folded state, the latter being indicative of at least one specific Ca(2+) binding pocket interfering directly with catalysis. Similar results are obtained with Mn(2+), Cd(2+), and [Co(NH(3))(6)](3+). Ni(2+) is a much more powerful inhibitor and the presence of either Zn(2+) or Pb(2+) 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 Mg(2+) and raises the question of biological relevance at least in the case of Ca(2+).
Institute of Inorganic Chemistry, University of Zurich, 8057 Zurich, Switzerland.
Michèle C
MC
Erat
Roland K O
RK
Sigel
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D002413:Cations, Divalent / Q000494:pharmacology*; D004272:DNA, Mitochondrial / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D047668:Inteins / Q000187:drug effects*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D019216:Metals, Heavy / Q000494:pharmacology; D012326:RNA Splicing / Q000187:drug effects*; D012329:RNA, Bacterial / Q000737:chemistry / Q000187:drug effects*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013997:Time Factors
D002413:Cations, Divalent; D004272:DNA, Mitochondrial; D019216:Metals, Heavy; D012329:RNA, Bacterial; D008274:Magnesium; D002118:Calcium