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Cellular Small Molecules Contribute to Twister Ribozyme Catalysis.

ABSTRACT: The number of self-cleaving small ribozymes has increased sharply in recent years. Advances have been made in describing these ribozymes in terms of four catalytic strategies: ? describes in-line attack, ? describes neutralization of the nonbridging oxygens, ? describes activation of the nucleophile, and ? describes stabilization of the leaving group. Current literature presents the rapid self-cleavage of the twister ribozyme in terms of all four of these classic catalytic strategies. Herein, we describe the nonspecific contribution of small molecules to ribozyme catalysis. At biological pH, the rate of the wild-type twister ribozyme is enhanced up to 5-fold in the presence of moderate buffer concentrations, similar to the 3-5-fold effects reported previously for buffer catalysis for protein enzymes. We observe this catalytic enhancement not only with standard laboratory buffers, but also with diverse biological small molecules, including imidazole, amino acids, and amino sugars. Brønsted plots suggest that small molecules assist in proton transfer, most likely with ? catalysis. Cellular small molecules provide a simple way to overcome the limited functional diversity of RNA and have the potential to participate in the catalytic mechanisms of many ribozymes in vivo.

SUBMITTER: Messina KJ

PROVIDER: S-EPMC6472948 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Cellular Small Molecules Contribute to Twister Ribozyme Catalysis.

Messina Kyle J KJ Bevilacqua Philip C PC

Journal of the American Chemical Society 20180813 33

The number of self-cleaving small ribozymes has increased sharply in recent years. Advances have been made in describing these ribozymes in terms of four catalytic strategies: α describes in-line attack, β describes neutralization of the nonbridging oxygens, γ describes activation of the nucleophile, and δ describes stabilization of the leaving group. Current literature presents the rapid self-cleavage of the twister ribozyme in terms of all four of these classic catalytic strategies. Herein, we ...[more]

PMID: 30102530

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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.

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Cellular Small Molecules Contribute to Twister Ribozyme Catalysis.

Publications

Cellular Small Molecules Contribute to Twister Ribozyme Catalysis.

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