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Metal Interactions with a GAAA RNA Tetraloop Characterized by 31P NMR and Phosphorothioate Substitutions

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journal contribution
posted on 22.06.2000, 00:00 by Melissa Maderia, Thomas E. Horton, Victoria J. DeRose
A metal site in a 5‘-GAAA-3‘ tetraloop, a stabilizing and phylogenetically conserved RNA motif, is explored using 31P NMR spectroscopy and phosphorothioate modifications. Similar to previous reports [Legault, P., and Pardi, A. (1994) J. Magn. Reson., Ser. B 103, 82−86], the 31P NMR spectrum of a 12-nucleotide stem−loop sequence 5‘-GGCCGAAAGGCC-3‘ exhibits resolved features from each of the phosphodiester linkages. Titration with Mg2+ results in distinct shifts of a subset of these 31P features, which are assigned to phosphodiesters 5‘ to A6, A7, and G5. Titration with Co(NH3)63+ causes only a slight upfield shift in the A6 feature, suggesting that changes caused by Mg2+ are due to inner-sphere metal−phosphate coordination. Rp-Phosphorothioate substitutions introduced enzymatically 5‘ to each of the three A residues of the tetraloop provide well-resolved 31P NMR features that are observed to shift in the presence of Cd2+ but not Mg2+, again consistent with a metal−phosphate site. Analysis of 31P NMR spectra using the sequence 5‘-GGGCGAAAGUCC-3‘ with single phosphorothioate substitutions in the loop region, separated into Rp and Sp diastereomers, provides evidence for an inner-sphere interaction with the phosphate 5‘ to A7 but outer-sphere or structural effects that cause perturbations 5‘ to A6. Introduction of an Rp-phosphorothioate 5‘ to A7 results in a distinct 31P NMR spectrum, consistent with thermodynamic studies reported in the accompanying paper that indicate a unique structure caused by this substitution. On the basis of these results and existing structural information, a metal site in the 5‘-GAAA-3‘ tetraloop is modeled using restrained molecular dynamics simulations.