title: Structure of the U6 RNA intramolecular stem-loop harboring an S(P)-phosphorothioate modification.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Reiter
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Nikstad
authors.forename: Anne M
authors.initials: AM
authors.lastname: Allmann
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Johnson
authors.forename: Samuel E
authors.initials: SE
authors.lastname: Butcher
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D008670:Metals / Q000378:metabolism; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017381:Spliceosomes / Q000378:metabolism; D013816:Thermodynamics; D013873:Thionucleotides / Q000737:chemistry
chemical_list: D008670:Metals; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D013873:Thionucleotides; C405486:U6 small nuclear RNA