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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.

Background: The peptide antibiotic viomycin inhibits ribosomal protein synthesis, group I intron self-splicing and self-cleavage of the human hepatitis delta virus ribozyme. To understand the molecular basis of RNA binding and recognition by viomycin, we isolated a variety of novel viomycin-binding RNA molecules using in vitro selection., Results: More than 90% of the selected RNA molecules shared one continuous highly conserved region of 14 nucleotides. Mutational analyses, structural probing, together with footprinting experiments by chemical modification, and Pb2+-induced cleavage showed that this conserved sequence harbours the antibiotic-binding site and forms a stem-loop structure. Moreover, the loop is engaged in a long-range interaction forming a pseudoknot., Conclusions: A comparison between the novel viomycin-binding motif and the natural RNA target sites for viomycin showed that all these segments form a pseudoknot at the antibiotic-binding site. We therefore conclude that this peptide antibiotic has a strong selectivity for particular RNA pseudoknots.

Self-splicing of group I introns requires divalent metal ions to promote catalysis as well as for the correct folding of the RNA. Lead cleavage has been used to probe the intron RNA for divalent metal ion binding sites. In the conserved core of the intron, only two sites of Pb2+ cleavage have been detected, which are located close to the substrate binding sites in the junction J8/7 and at the bulged nucleotide in the P7 stem. Both lead cleavages can be inhibited by high concentrations of Mg2+ and Mn2+ ions, suggesting that they displace Pb2+ ions from the binding sites. The RNA is protected from lead cleavage by 2'-deoxyGTP, a competitive inhibitor of splicing. The two major lead induced cleavages are both located in the conserved core of the intron and at phosphates, which had independently been demonstrated to interact with magnesium ions and to be essential for splicing. Thus, we suggest that the conditions required for lead cleavage occur mainly at those sites, where divalent ions bind that are functionally involved in catalysis. We propose lead cleavage analysis of functional RNA to be a useful tool for mapping functional magnesium ion binding sites.