Nick Reiter
Last active: 1/21/2015

Structure of the U6 RNA intramolecular stem-loop harboring an S(P)-phosphorothioate modification.

Reiter NJ, Nikstad LJ, Allmann AM, Johnson RJ, Butcher SE
RNA. 2003 9 (5): 533-42

PMID: 12702812 · PMCID: PMC1370419 · DOI:10.1261/rna.2199103

Phosphorothioate-substitution experiments are often used to elucidate functionally important metal ion-binding sites on RNA. All previous experiments with S(P)-phosphorothioate-substituted RNAs have been done in the absence of structural information for this particular diastereomer. Yeast U6 RNA contains a metal ion-binding site that is essential for spliceosome function and includes the pro-S(P) oxygen 5' of U(80). S(P)-phosphorothioate substitution at this location creates spliceosomes dependent on thiophilic ions for the first step of splicing. We have determined the solution structure of the U(80) S(P)-phosphorothioate-substituted U6 intramolecular stem-loop (ISL), and also report the refined NMR structure of the unmodified U6 ISL. Both structures were determined with inclusion of (1)H-(13)C residual dipolar couplings. The precision of the structures with and without phosphorothioate (RMSD = 1.05 and 0.79 A, respectively) allows comparison of the local and long-range structural effect of the modification. We find that the U6-ISL structure is unperturbed by the phosphorothioate. Additionally, the thermodynamic stability of the U6 ISL is dependent on the protonation state of the A(79)-C(67) wobble pair and is not affected by the adjacent phosphorothioate. These results indicate that a single S(P)-phosphorothioate substitution can be structurally benign, and further validate the metal ion rescue experiments used to identify the essential metal-binding site(s) in the spliceosome.

MeSH Terms (12)

Base Sequence Binding Sites Metals Models, Molecular Nuclear Magnetic Resonance, Biomolecular Nucleic Acid Conformation RNA, Fungal RNA, Small Nuclear Saccharomyces cerevisiae Spliceosomes Thermodynamics Thionucleotides

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