Journal
BIOCHEMISTRY
Volume 39, Issue 28, Pages 8193-8200Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi000140l
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Funding
- NIGMS NIH HHS [GM58096, R01 GM058096-02, R01 GM058096] Funding Source: Medline
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A metal site in a 5'-GAAA-3' tetraloop, a stabilizing and phylogenetically conserved RNA motif, is explored using P-31 NMR spectroscopy and phosphorothioate 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 12-nucleotide stem-loop sequence 5'-GGCC<(GAA)under bar>GGCC-3' exhibits resolved 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 coordination. R-p-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 P-31 NMR spectra using the sequence 5'-GGGC<(GAAA)under bar>GUCC-3' with single phosphorothioate substitutions 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 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.
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