201. Visualizing metal-ion-binding sites in group I introns by iron(II)-mediated Fenton reactions.
- Author
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Berens C, Streicher B, Schroeder R, and Hillen W
- Subjects
- Animals, Base Sequence, Binding Sites, Binding, Competitive, Cations, Divalent, Crystallography, X-Ray, Hydrolysis, Molecular Sequence Data, Nucleic Acid Conformation, RNA, Catalytic chemistry, RNA, Protozoan chemistry, Ribonucleotide Reductases genetics, Ferrous Compounds chemistry, Introns, Metals metabolism, RNA, Catalytic metabolism, RNA, Protozoan metabolism
- Abstract
Background: Most catalytic RNAs depend on divalent metal ions for folding and catalysis. A thorough structure-function analysis of catalytic RNA therefore requires the identification of the metal-ion-binding sites. Here, we probed the binding sites using Fenton chemistry, which makes use of the ability of Fe2+ to functionally or structurally replace Mg2+ at ion-binding sites and to generate short-lived and highly reactive hydroxyl radicals that can cleave nucleic acid and protein backbones in spatial proximity of these ion-binding sites., Results: Incubation of group I intron RNA with Fe2+, sodium ascorbate and hydrogen peroxide yields distinctly cleaved regions that occur only in the correctly folded RNA in the presence of Mg2+ and can be competed by additional Mg2+, suggesting that Fe2+ and Mg2+ interact with the same sites. Cleaved regions in the catalytic core are conserved for three different group I introns, and there is good correlation between metal-ion-binding sites determined using our method and those determined using other techniques. In a model of the T4 phage-derived td intron, cleaved regions separated in the secondary structure come together in three-dimensional space to form several metal-ion-binding pockets., Conclusions: In contrast to structural probing with Fe2+/EDTA, cleavage with Fe2+ detects metal-ion-binding sites located primarily in the inside of the RNA. Essentially all metal-ion-binding pockets detected are formed by tertiary structure elements. Using this method, we confirmed proposed metal-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.
- Published
- 1998
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