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- Title
A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.
- Authors
Košutić, Marija; Neuner, Sandro; Ren, Aiming; Flür, Sara; Wunderlich, Christoph; Mairhofer, Elisabeth; Vušurović, Nikola; Seikowski, Jan; Breuker, Kathrin; Höbartner, Claudia; Patel, Dinshaw J.; Kreutz, Christoph; Micura, Ronald
- 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 13C2-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 Mg2+ 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 Mn2+ or Cd2+ 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.
- Subjects
CATALYTIC RNA; CLEAVAGE (Embryology); PHOSPHODIESTERS; PHYLOGENY; MUTAGENESIS; NUCLEAR magnetic resonance spectroscopy; ADENINE
- Publication
Angewandte Chemie International Edition, 2015, Vol 54, Issue 50, p15128
- ISSN
1433-7851
- Publication type
Article
- DOI
10.1002/anie.201506601