VISUALIZING METAL-ION-BINDING SITES IN GROUP-I INTRONS BY IRON(II)-MEDIATED FENTON REACTIONS

Background: Most catalytic RNAs depend on divalent metal ions for fold ing and catalysis, A thorough structure-function analysis of catalytic RNA therefore requires the identification of the metal-ion-binding si tes, Here, we probed the binding sites using Fenton chemistry, which m akes use of the ability of Fe2+ to functionally or structurally replac e Mg2+ at ion-binding sites and to generate short-lived and highly rea ctive hydroxyl radicals that can cleave nucleic acid and protein backb ones in spatial proximity of these ion-binding sites. Results: Incubat ion of group I intron RNA with Fe2+, sodium ascorbate and hydrogen per oxide yields distinctly cleaved regions that occur only in the correct ly folded RNA in the presence of Mg2+ and can be competed by additiona l Mg2+, suggesting that Fe2+ and Mg2+ interact with the same sites, Cl eaved regions in the catalytic core are conserved for three different group I introns, and there is good correlation between metal-ion-bindi ng sites determined using our method and those determined using other techniques. In a model of the T4 phage-derived td intron, cleaved regi ons separated in the secondary structure come together in three-dimens ional space to form several metal-ion-binding pockets. Conclusions: In contrast to structural probing with Fe2+/EDTA, cleavage with Fe2+ det ects metal-ion-binding sites located primarily in the inside of the RN A, Essentially all metal-ion-binding pockets detected are formed by te rtiary structure elements, Using this method, we confirmed proposed me tal-ion-binding sites and identified new ones in group I intron RNAs, This approach should allow the localization of metal-ion-binding sites in RNAs of interest.