Dataset Information

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.

SUBMITTER: Kosutic M

PROVIDER: S-EPMC4715771 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

Košutić Marija M Neuner Sandro S Ren Aiming A Flür Sara S Wunderlich Christoph C Mairhofer Elisabeth E Vušurović Nikola N Seikowski Jan J Breuker Kathrin K Höbartner Claudia C Patel Dinshaw J DJ Kreutz Christoph C Micura Ronald R

Angewandte Chemie (International ed. in English) 20151016 50

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 signifi ...[more]

PMID: 26473980

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Project description:We present results from molecular dynamics simulations and free energy calculations of the twister ribozyme at different stages along the reaction path to gain insight into its mechanism. The results, together with recent biochemical experiments, provide support for a mechanism involving general-acid catalysis by a conserved adenine residue in the active site. Although adenine has been previously implicated as a general acid acting through the N1 position in other ribozymes such as the hairpin and VS ribozymes, in the twister ribozyme there may be a twist. Biochemical experiments suggest that general acid catalysis may occur through the N3 position, which has never before been implicated in this role; however, currently, there is a lack of a detailed structural model for the active state of the twister ribozyme in solution that is consistent with these and other experiments. Simulations in a crystalline environment reported here are consistent with X-ray crystallographic data, and suggest that crystal packing contacts trap the RNA in an inactive conformation with U-1 in an extruded state that is incompatible with an in-line attack to the scissile phosphate. Simulations in solution, on the other hand, reveal this region to be dynamic and able to adopt a conformation where U-1 is stacked with G33. In this state, the nucleophile is in line with the scissile phosphate, and the N1 position of G33 and N3 position of A1 are poised to act as a general base and acid, respectively, as supported by mutational experiments. Free energy calculations further predict the electrostatic environment causes a shift of the microscopic pKa at the N3 position of A1 toward neutrality by approximately 5 pKa units. These results offer a unified interpretation of a broad range of currently available experimental data that points to a novel mode of general acid catalysis through the N3 position of an adenine nucleobase, thus expanding the repertoire of known mechanistic strategies employed by small nucleolytic ribozymes.

Dataset Information

A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

Publications

A Mini-Twister Variant and Impact of Residues/Cations on the Phosphodiester Cleavage of this Ribozyme Class.

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