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Use of luminescence resonance energy transfer to identify locations of site-bound metal ions in the spliceosomal U2–U6 snRNA complex
U2 and U6 snRNAs pair to form a phylogenetically conserved complex at the catalytic core of the spliceosome. Interactions with divalent metal ions, particularly Mg(II), at specific sites are essential for its structural stability, folding and catalytic activity. We used a new Förster Resonance Energy Transfer (FRET) method between site- bound luminescent lanthanide ions and a covalently attached fluorescent dye, as well as supporting stoichiometric and mutational studies, to determine the locations of site-bound Tb(III) on the U2-U6 complex. At pH 7.2, we detected three metal ion binding sites situated in: 1) the consensus ACACAGA sequence, which forms the internal loop between helices I and III; 2) the four-way-junction, which contains the conserved AGC triad, and; 3) the internal loop of the U6 intra-molecular stem loop (ISL). Binding at each of these sites is supported by previous phosphorothioate substitution studies and, in the case of the ISL site, by NMR-based structural studies. Binding of Tb(III) at the four-way junction and the ISL sites was found to be pH-dependent, with no ion binding observed below pH 6 and 7, respectively. This pH-dependence of metal ion binding suggests that the local environment may play a role in the binding of metal ions, which, in turn, regulates splicing activity.
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