title: A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.
abstract: Nucleolytic ribozymes catalyze site-specific cleavage of their phosphodiester backbones. A minimal version of the twister ribozyme is reported that lacks the phylogenetically conserved stem P1 while retaining wild-type activity. Atomic mutagenesis revealed that nitrogen atoms N1 and N3 of the adenine-6 at the cleavage site are indispensable for cleavage. By NMR spectroscopy, a pKa value of 5.1 was determined for a (13) C2-labeled adenine at this position in the twister ribozyme, which is significantly shifted compared to the pKa of the same adenine in the substrate alone. This finding pinpoints at a potential role for adenine-6 in the catalytic mechanism besides the previously identified invariant guanine-48 and a Mg(2+) ion, both of which are directly coordinated to the non-bridging oxygen atoms of the scissile phosphate; for the latter, additional evidence stems from the observation that Mn(2+) or Cd(2+) accelerated cleavage of phosphorothioate substrates. The relevance of this metal ion binding site is further emphasized by a new 2.6 Å X-ray structure of a 2'-OCH3 -U5 modified twister ribozyme.
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck (Austria).
authors.forename: Marija
authors.initials: M
authors.lastname: Košutić
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck (Austria).
authors.forename: Sandro
authors.initials: S
authors.lastname: Neuner
authors.affiliation: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, NY, New York 10065 (USA).
authors.forename: Aiming
authors.initials: A
authors.lastname: Ren
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck (Austria).
authors.forename: Sara
authors.initials: S
authors.lastname: Flür
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck (Austria).
authors.forename: Christoph
authors.initials: C
authors.lastname: Wunderlich
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck (Austria).
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Mairhofer
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck (Austria).
authors.forename: Nikola
authors.initials: N
authors.lastname: Vušurović
authors.affiliation: Max Planck Institute of Biophysical Chemistry and Institute of Organic and Biomolecular Chemistry, Georg August University of Göttingen, 37077 Göttingen (Germany).
authors.forename: Jan
authors.initials: J
authors.lastname: Seikowski
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck (Austria).
authors.forename: Kathrin
authors.initials: K
authors.lastname: Breuker
authors.affiliation: Max Planck Institute of Biophysical Chemistry and Institute of Organic and Biomolecular Chemistry, Georg August University of Göttingen, 37077 Göttingen (Germany).
authors.forename: Claudia
authors.initials: C
authors.lastname: Höbartner
authors.affiliation: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, NY, New York 10065 (USA).
authors.forename: Dinshaw J
authors.initials: DJ
authors.lastname: Patel
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck (Austria).
authors.forename: Christoph
authors.initials: C
authors.lastname: Kreutz
authors.affiliation: Institute of Organic Chemistry and Center for Molecular Biosciences CMBI, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck (Austria).
authors.forename: Ronald
authors.initials: R
authors.lastname: Micura
mesh_terms: D000225:Adenine / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D002104:Cadmium / Q000737:chemistry / Q000378:metabolism; D002412:Cations / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010755:Organophosphates / Q000737:chemistry* / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*
chemical_list: D002412:Cations; D010755:Organophosphates; D016337:RNA, Catalytic; D002104:Cadmium; D008345:Manganese; D000225:Adenine
keywords: metal ion rescue; nucleoside modifications; oligoribonucleotides; perturbed pKa; solid-phase synthesis