Engineering a ribozyme with aminoacyl-tRNA synthetase activity

bioRxiv (Cold Spring Harbor Laboratory) · 2024

• Chemistry
• Computational biology
• Biochemistry

A ribozyme that can charge a tRNA with amino acids and discriminate between cognate and non-cognate tRNAs is of interest because an RNA with this ability may have been a critical for translation in the transition from the RNA world. In addition, it could provide a tool for incorporating non-canonical amino acids for biotechnology applications. Here, we rationally engineer a ribozyme by fusing a tRNA binding module derived from a T-box riboswitch with a catalytic module (a flexizyme) to generate a ribozyme that can amino acylate a target tRNA. We demonstrate that this ribozyme be readily redesigned to alter tRNA specificity. This ribozyme is compatible with an in vitro translation system and could be used to recode a protein sequence to site-specifically incorporate a non-canonical amino acid.

RNA | Catalysis by RNA, Structural Themes: Group I Introns

Elsevier eBooks · 2021

• Biology
• Genetics
• Molecular biology

RNA-Puzzles Round IV: 3D structure predictions of four ribozymes and two aptamers

RNA · 2020 · 172 citations

• Computational biology
• Biology
• Genetics

RNA-Puzzles is a collective endeavor dedicated to the advancement and improvement of RNA 3D structure prediction. With agreement from crystallographers, the RNA structures are predicted by various groups before the publication of the crystal structures. We now report the prediction of 3D structures for six RNA sequences: four nucleolytic ribozymes and two riboswitches. Systematic protocols for comparing models and crystal structures are described and analyzed. In these six puzzles, we discuss (i) the comparison between the automated web servers and human experts; (ii) the prediction of coaxial stacking; (iii) the prediction of structural details and ligand binding; (iv) the development of novel prediction methods; and (v) the potential improvements to be made. We show that correct prediction of coaxial stacking and tertiary contacts is essential for the prediction of RNA architecture, while ligand binding modes can only be predicted with low resolution and simultaneous prediction of RNA structure with accurate ligand binding still remains out of reach. All the predicted models are available for the future development of force field parameters and the improvement of comparison and assessment tools.

Faculty Opinions recommendation of Cryo-EM Structure of the Human Ribonuclease P Holoenzyme.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature · 2019-01-07

Faculty Opinions recommendation of Structural basis for tRNA decoding and aminoacylation sensing by T-box riboregulators.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature · 2019-11-22

Divalent Metal Ion Activation of a Guanine General Base in the Hammerhead Ribozyme: Insights from Molecular Simulations

Biochemistry · 2017-05-22 · 54 citations

The hammerhead ribozyme is a well-studied nucleolytic ribozyme that catalyzes the self-cleavage of the RNA phosphodiester backbone. Despite experimental and theoretical efforts, key questions remain about details of the mechanism with regard to the activation of the nucleophile by the putative general base guanine (G12). Straightforward interpretation of the measured activity–pH data implies the pKa value of the N1 position in the G12 nucleobase is significantly shifted by the ribozyme environment. Recent crystallographic and biochemical work has identified pH-dependent divalent metal ion binding at the N7/O6 position of G12, leading to the hypothesis that this binding mode could induce a pKa shift of G12 toward neutrality. We present computational results that support this hypothesis and provide a model that unifies the interpretation of available structural and biochemical data and paints a detailed mechanistic picture of the general base step of the reaction. Experimentally testable predictions are made for mutational and rescue effects on G12, which will give further insights into the catalytic mechanism. These results contribute to our growing knowledge of the potential roles of divalent metal ions in RNA catalysis.

Two Active Site Divalent Ions in the Crystal Structure of the Hammerhead Ribozyme Bound to a Transition State Analogue

Biochemistry · 2015-11-09 · 73 citations

The crystal structure of the hammerhead ribozyme bound to the pentavalent transition state analogue vanadate reveals significant rearrangements relative to the previously determined structures. The active site contracts, bringing G10.1 closer to the cleavage site and repositioning a divalent metal ion such that it could, ultimately, interact directly with the scissile phosphate. This ion could also position a water molecule to serve as a general acid in the cleavage reaction. A second divalent ion is observed coordinated to O6 of G12. This metal ion is well-placed to help tune the pKA of G12. On the basis of this crystal structure as well as a wealth of biochemical studies, we propose a mechanism in which G12 serves as the general base and a magnesium-bound water serves as a general acid.

Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

Biochemistry · 2015-09-23 · 68 citations

The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn(2+) specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH-rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction.

Two divalent metal ions and conformational changes play roles in the hammerhead ribozyme cleavage reaction-G12A mutant in Mg2+

2015-08-29

Two divalent metal ions and conformational changes play roles in the hammerhead ribozyme cleavage reaction-WT ribozyme in Mn2+ at high pH

2015-08-31