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Title
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Structure of the U6 RNA intramolecular stem-loop harboring an S(P)-phosphorothioate modification.
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Authors
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N.J.Reiter,
L.J.Nikstad,
A.M.Allmann,
R.J.Johnson,
S.E.Butcher.
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Ref.
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Rna, 2003,
9,
533-542.
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PubMed id
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Abstract
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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.
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