Metal interactions with a GAAA RNA tetraloop characterized by P-31 NMR andphosphorothioate substitutions

A metal site in a 5'-GAAA-3' tetraloop, a stabilizing and phylogenetically conserved RNA motif, is explored using P-31 NMR spectroscopy and phosphorot hioate modifications. Similar to previous reports [Legault, P., and Pardi, A. (1994) J. Magn. Reson., Ser. B 103, 82-86], the P-31 NMR spectrum of a 1 2-nucleotide stem-loop sequence 5'-GGCC<(GAA)under bar>GGCC-3' exhibits res olved features from each of the phosphodiester linkages. Titration with Mg2 + results in distinct shifts of a subset of these P-3 1 features, which are assigned to phosphodiesters 5' to A6, A7, and 05. Titration with Co(NH3)(6 )(3+) causes only a slight upfield shift in the A6 feature, suggesting that changes caused by Mg2+ are due to inner-sphere metal-phosphate coordinatio n. R-p-Phosphorothioate substitutions introduced enzymatically 5' to each o f the three A residues of the tetraloop provide well-resolved 31P NMR featu res that are observed to shift in the presence of Cd2+ but not Mg2+, again consistent with a metal-phosphate site. Analysis of P-31 NMR spectra using the sequence 5'-GGGC<(GAAA)under bar>GUCC-3' with single phosphorothioate s ubstitutions in the loop region, separated into R-p and S-p 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 R-p-phosphorothioate 5' to A7 results in a distinct P- 31 NMR spectrum, consistent with thermodynamic studies reported in the acco mpanying paper that indicate a unique structure caused by this substitution . On the basis of these results and existing structural information, a meta l site in the 5'-GAAA-3' tetraloop is modeled using restrained molecular dy namics simulations.