<i>Who stole the proton?</i> Suspect general base guanine found with a smoking gun in the pistol ribozyme

Ekesan, Şölen; York, Darrin M.

doi:10.1039/d2ob00234e

Cited by 5 publications

(5 citation statements)

References 87 publications

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“…We have previously reported computational simulations for the HHr, Psr, , 8–17dz, and VSr systems, and also made cross-cutting studies of the role of the divalent metal ion in promotion of productive hydrogen bonding of the O2′ nucleophile to facilitate its activation . The details of the system setup, convergence of the simulations, and other specialized analysis can be found in these other works.…”

Section: Methodsmentioning

confidence: 99%

RNA Electrostatics: How Ribozymes Engineer Active Sites to Enable Catalysis

et al. 2022

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Electrostatic interactions are fundamental to RNA structure and function, and intimately influenced by solvation and the ion atmosphere. RNA enzymes, or ribozymes, are catalytic RNAs that are able to enhance reaction rates over a million-fold, despite having only a limited repertoire of building blocks and available set of chemical functional groups. Ribozyme active sites usually occur at junctions where negatively charged helices come together, and in many cases leverage this strained electrostatic environment to recruit metal ions in solution that can assist in catalysis. Similar strategies have been implicated in related artificially engineered DNA enzymes. Herein, we apply Poisson–Boltzmann, 3D-RISM, and molecular simulations to study a set of metal-dependent small self-cleaving ribozymes (hammerhead, pistol, and Varkud satellite) as well as an artificially engineered DNAzyme (8–17) to examine electrostatic features and their relation to the recruitment of monovalent and divalent metal ions important for activity. We examine several fundamental roles for these ions that include: (1) structural integrity of the catalytically active state, (2) pK a tuning of residues involved in acid–base catalysis, and (3) direct electrostatic stabilization of the transition state via Lewis acid catalysis. Taken together, these examples demonstrate how RNA electrostatics orchestrates the site-specific and territorial binding of metal ions to play important roles in catalysis.

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Section: Methodsmentioning

confidence: 99%

RNA Electrostatics: How Ribozymes Engineer Active Sites to Enable Catalysis

et al. 2022

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“…To gain further insights into the Dz's mode of action, future MD studies may consider (a) the use of molecular solvation theory to predict water and metal ion binding sites in nucleic acids [47] or (b) treating the catalytic centre at the quantum mechanical level while using a molecular mechanics representation of the rest of the system or (c) using a deprotonated catalytic base to represent the state of attack better [30,31,48]. However, these methods generally work best in conjunction with an X-ray crystal structure of the DNAzyme that includes co-crystallised metal ions at the catalytic centre.…”

Section: Catalysismentioning

confidence: 99%

The architecture of the 10‐23 DNAzyme and its implications for DNA‐mediated catalysis

et al. 2022

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Understanding the molecular features of catalytically active DNA sequences, so‐called DNAzymes, is essential not only for our understanding of the fundamental properties of catalytic nucleic acids in general, but may well be the key to unravelling their full potential via tailored modifications. Our recent findings contributed to the endeavour to assemble a mechanistic picture of DNA‐mediated catalysis by providing high‐resolution structural insights into the 10‐23 DNAzyme (Dz) and exposing a complex interplay between the Dz's unique molecular architecture, conformational plasticity, and dynamic modulation by metal ions as central elements of the DNA catalyst. Here, we discuss key features of our findings and compare them to other studies on similar systems.

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“… [60] Under the assumption of a local rearrangement (into a L‐platform/L‐scaffold framework) simulated by molecular dynamics calculations, G42 has been proposed to act as general base in a primary catalytic pathway. [60] Having the c 1 G modification in hands, we set out to experimentally test this hypothesis with the corresponding G42c 1 G mutant. We found that the cleavage rate for the mutated ribozyme was 0.89±0.06 min −1 which is only 4.2‐fold slower compared to the wildtype ribozyme (Supporting Figure S11, Supporting Table S3).…”

Section: Resultsmentioning

confidence: 99%

Scaling Catalytic Contributions of Small Self‐Cleaving Ribozymes

et al. 2022

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Nucleolytic ribozymes utilize general acid-base catalysis to perform phosphodiester cleavage. In most ribozyme classes, a conserved active site guanosine is positioned to act as general base, thereby activating the 2'-OH group to attack the scissile phosphate (γ-catalysis). Here, we present an atomic mutagenesis study for the pistol ribozyme class. Strikingly, "general base knockout" by replacement of the guanine N1 atom by carbon results in only 2.7-fold decreased rate. Therefore, the common view that γ-catalysis critically depends on the N1 moiety becomes challenged. For pistol ribozymes we found that γ-catalysis is subordinate in overall catalysis, made up by two other catalytic factors (α and δ). Our approach allows scaling of the different catalytic contributions (α, β, γ, δ) with unprecedented precision and paves the way for a thorough mechanistic understanding of nucleolytic ribozymes with active site guanines.

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Who stole the proton? Suspect general base guanine found with a smoking gun in the pistol ribozyme

Abstract: The pistol ribozyme (Psr) is one among the most recently discovered classes of small nucleolytic ribozymes that catalyze site-specific RNA self-cleavage through 2′-O-transphosphorylation. The Psr contains a conserved guanine (G40)...

Cited by 5 publications

References 87 publications

RNA Electrostatics: How Ribozymes Engineer Active Sites to Enable Catalysis

RNA Electrostatics: How Ribozymes Engineer Active Sites to Enable Catalysis

The architecture of the 10‐23 DNAzyme and its implications for DNA‐mediated catalysis

Scaling Catalytic Contributions of Small Self‐Cleaving Ribozymes

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