A mini-twister variant and impact of residues/cations on the phosphodiester cleavage of this ribozyme class Journal Article
| Authors: | Košutic, M.; Neuner, S.; Ren, A.; Flür, S.; Wunderlich, C.; Mairhofer, E.; Vušurovic, N.; Seikowski, J.; Breuker, K.; Höbartner, C.; Patel, D. J.; Kreutz, C.; Micura, R. |
| Article 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 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. Efficient cleavage of the twister ribozyme class does not require the phylogenetically conserved stem P1. The shifted pKa of the adenine at the cleavage site together with Mn2+- or Cd2+-accelerated cleavage of SP phosphorothioate substrates suggest the combination of nucleobase-assisted acid-base catalysis and metal-ion catalysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. |
| Keywords: | nuclear magnetic resonance spectroscopy; synthesis (chemical); substrates; metals; esters; solid-phase synthesis; solid phase synthesis; manganese; catalytic mechanisms; oligoribonucleotides; metal ions; metal ion rescue; nucleoside modifications; perturbed pka; atoms; metal ion binding; non-bridging oxygen atoms; perturbed pk<sub>a</sub>; phosphorothioates; cadmium compounds |
| Journal Title: | Angewandte Chemie - International Edition |
| Volume: | 54 |
| Issue: | 50 |
| ISSN: | 1433-7851 |
| Publisher: | Wiley Blackwell |
| Date Published: | 2015-12-07 |
| Start Page: | 15128 |
| End Page: | 15133 |
| Language: | English |
| DOI: | 10.1002/anie.201506601 |
| PROVIDER: | scopus |
| PMCID: | PMC4715771 |
| PUBMED: | 26473980 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 3 February 2016 -- Source: Scopus |
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