Your browser doesn't support javascript.
loading
Specific phosphorothioate substitution within domain 6 of a group II intron ribozyme leads to changes in local structure and metal ion binding.
Erat, Michèle C; Besic, Emina; Oberhuber, Michael; Johannsen, Silke; Sigel, Roland K O.
Afiliación
  • Erat MC; Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
  • Besic E; Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
  • Oberhuber M; Institute of Organic Chemistry, University of Innsbruck, Innrain 52a, 6020, Innsbruck, Austria.
  • Johannsen S; Laimburg Research Center, Laimburg 6 Pfatten (Vadena), 39040, Auer (Ora), BZ, Italy.
  • Sigel RKO; Department of Chemistry, University of Zürich, Winterthurerstrasse 190, 8057, Zurich, Switzerland. silke.johannsen@chem.uzh.ch.
J Biol Inorg Chem ; 23(1): 167-177, 2018 01.
Article en En | MEDLINE | ID: mdl-29218637
Group II introns are large self-splicing ribozymes that require high amounts of monovalent and divalent metal ions for folding and catalysis under in vitro conditions. Domain 6 of these ribozymes contains a highly conserved adenosine whose 2'-OH acts as a nucleophile during self-cleavage via the branching pathway. We have previously suggested a divalent metal ion that binds to the major groove at the GU wobble pair above the branch-A in a minimal, but active branch domain construct (D6-27) from the yeast mitochondrial intron Sc.ai5γ. Here we characterize metal ion binding to the phosphate oxygens at the branch site. In vitro transcription yielded a D6-27 construct where all R P oxygens of the uridine phosphate groups are replaced by sulfur (α-thio-D6-27). We determined its NMR structure, the second RNA-only structure containing thiophosphate groups. [31P] resonances were assigned and chemical shift changes monitored upon titration with Cd2+. In addition, the two uridines flanking the branch-point, U19 and U21 were specifically thioated by chemical synthesis (thio-U19-D6-27 and thio-U19/U21-D6-27), enabling us to study Cd2+ binding at the R P-, as well as the S P- position of the corresponding phosphate oxygens. Our studies reveal that both non-bridging phosphate oxygens of U19 are involved in metal ion coordination, whereas only the major groove phosphate oxygen of U21 is influenced. Together with NOE data of a hexaamminecobalt(III) titration, this suggests a single metal ion binding site at the GU wobble pair above the branch point in the major groove of D6 of this group II intron ribozyme.
Asunto(s)
Palabras clave

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Organotiofosfatos / Cadmio / Intrones / ARN Catalítico / Complejos de Coordinación / Magnesio Idioma: En Revista: J Biol Inorg Chem Asunto de la revista: BIOQUIMICA Año: 2018 Tipo del documento: Article País de afiliación: Suiza Pais de publicación: Alemania

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Organotiofosfatos / Cadmio / Intrones / ARN Catalítico / Complejos de Coordinación / Magnesio Idioma: En Revista: J Biol Inorg Chem Asunto de la revista: BIOQUIMICA Año: 2018 Tipo del documento: Article País de afiliación: Suiza Pais de publicación: Alemania