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Journal Article

A mini-twister variant and impact of residues/cations on the phosphodiester cleavage of this ribozyme class.

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Seikowski,  J.
Research Group of Nucleic Acid Chemistry, MPI for Biophysical Chemistry, Max Planck Society;

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Höbartner,  C.
Research Group of Nucleic Acid Chemistry, MPI for Biophysical Chemistry, Max Planck Society;

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Citation

Kosutic, M., Neuner, S., Ren, A., Flür, S., Wunderlich, C., Mairhofer, E., et al. (2015). A mini-twister variant and impact of residues/cations on the phosphodiester cleavage of this ribozyme class. Angewandte Chemie International Edition, 54(50), 15128-15133. doi:10.1002/anie.201506601.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0029-713B-A
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.