Dataset Information

Small Molecule Rescue and Glycosidic Conformational Analysis of the Twister Ribozyme.

ABSTRACT: The number of self-cleaving ribozymes has increased sharply in recent years, giving rise to elaborations of the four known ribozyme catalytic strategies, ?, ?, ?, and ?. One such extension is utilized by the twister ribozyme, which is hypothesized to conduct ?, or general acid catalysis, via N3 of the syn adenine +1 nucleobase indirectly via buffer catalysis at biological pH and directly at lower pH. Herein, we test the ? catalysis role of A1 via chemical rescue and the catalytic relevance of the syn orientation of the nucleobase by conformational analysis. Using inhibited twister ribozyme variants with A1(N3) deaza or A1 abasic modifications, we observe >100-fold chemical rescue effects in the presence of protonatable biological small molecules such as imidazole and histidine, similar to observed rescue values previously reported for C75U/C76? in the HDV ribozyme. Brønsted plots for the twister variants support a model in which small molecules rescue catalytic activity via a proton transfer mechanism, suggesting that A1 in the wild type is involved in proton transfer, most likely general acid catalysis. Additionally, through glycosidic conformational analysis in an appropriate background that accommodates the bromine atom, we observe that an 8BrA1-modified twister ribozyme is up to 10-fold faster than a nonmodified A1 ribozyme, supporting crystallographic data that show that A1 is syn when conducting proton transfer. Overall, this study provides functional evidence that the nucleotide immediately downstream of the cleavage site participates directly or indirectly in general acid-base catalysis in the twister ribozyme while occupying the syn conformation.

SUBMITTER: Messina KJ

PROVIDER: S-EPMC6901379 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Small Molecule Rescue and Glycosidic Conformational Analysis of the Twister Ribozyme.

Messina Kyle J KJ Kierzek Ryszard R Tracey Matthew A MA Bevilacqua Philip C PC

Biochemistry 20191119 48

The number of self-cleaving ribozymes has increased sharply in recent years, giving rise to elaborations of the four known ribozyme catalytic strategies, α, β, γ, and δ. One such extension is utilized by the twister ribozyme, which is hypothesized to conduct δ, or general acid catalysis, via N3 of the <i>syn</i> adenine +1 nucleobase indirectly via buffer catalysis at biological pH and directly at lower pH. Herein, we test the δ catalysis role of A1 via chemical rescue and the catalytic relevanc ...[more]

PMID: 31742390

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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

Small Molecule Rescue and Glycosidic Conformational Analysis of the Twister Ribozyme.

Publications

Small Molecule Rescue and Glycosidic Conformational Analysis of the Twister Ribozyme.

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