Ribozyme Structures and Mechanisms

Doherty, Elizabeth; Doudna, Jennifer A.

doi:10.1146/annurev.biophys.30.1.457

Cited by 192 publications

(132 citation statements)

References 143 publications

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“…In the former, the reaction coordinate is defined as (2) where R P-O 5′ and R P-O 2′ are the distances of the ribosyl O 5′ and O 5′ atoms from the P atom, respectively. When two reaction coordinates are considered, the first reaction coordinate ζ 1 is the same as that of eq 2, and the second reaction coordinate ζ 2 is defined as (3) where R Donor-H and R Acceptor-H are the distances of the proton to be transferred from the donor and to the acceptor atom, respectively. The one-dimensional PMF models the free energy change for the nucleophilic substitution reaction itself, whereas the two-dimensional PMF addresses the question of concerted versus stepwise mechanism involving both substitution and proton transfer in a general acid-base catalysis.…”

Section: Free Energy Simulationsmentioning

confidence: 99%

Quantum Mechanical/Molecular Mechanical Simulation Study of the Mechanism of Hairpin Ribozyme Catalysis

2008

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The molecular mechanism of hairpin ribozyme catalysis is studied with molecular dynamics simulations using a combined quantum mechanical and molecular mechanical (QM/MM) potential with a recently developed semiempirical AM1/d-PhoT model for phosphoryl transfer reactions. Simulations are used to derive one-and two-dimensional potentials of mean force to examine specific reaction paths and assess the feasibility of proposed general acid and base mechanisms. Densityfunctional calculations of truncated active site models provide complementary insight to the simulation results. Key factors utilized by the hairpin ribozyme to enhance the rate of transphosphorylation are presented, and the roles of A38 and G8 as general acid and base catalysts are discussed. The computational results are consistent with available experimental data, provide support for a general acid/base mechanism played by functional groups on the nucleobases, and offer important insight into the ability of RNA to act as a catalyst without explicit participation by divalent metal ions.

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Section: Free Energy Simulationsmentioning

confidence: 99%

Quantum Mechanical/Molecular Mechanical Simulation Study of the Mechanism of Hairpin Ribozyme Catalysis

2008

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“…This creates a nucleophile of sufficient strength to attack the phosphorous of an adjacent or nearby phosphate group. 16 A "patch" 42 has been prepared for the standard CHARMM27 RNA nucleotide which removes the 2′OH hydrogen and modifies the 2′O − charge, L-J interactions and geometry of nearby atoms. Parameterization of this patch is discussed below.…”

Section: ′O Deprotonated Sugar Ringmentioning

confidence: 99%

“…A common biological reaction catalyzed by several prototype ribozyme systems, such as the hammerhead, 10,11 hairpin, 12,13 and hepatitis delta virus 14,15 ribozymes, involves cleavage of a phosphate group through a transesterification reaction 16,17 (Figure 1). In this reaction, the 2′ OH of the ribose sugar ring becomes activated via deprotonation and makes an in-line attack to the adjacent 3′-phosphate along the phosphodiester backbone.…”

Section: Introductionmentioning

confidence: 99%

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

et al. 2006

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Force field parameters specifically optimized for residues important in the study of RNA catalysis are derived from density-functional calculations in a fashion consistent with the CHARMM27 allatom empirical force field. Parameters are presented for residues that model reactive RNA intermediates and transition state analogs, thio-substituted phosphates and phosphoranes, and bound Mg 2+ and di-metal bridge complexes. Target data was generated via density-functional calculations at the B3LYP/6-311++G(3df,2p)//B3LYP/6-31++G(d,p) level. Partial atomic charges were initially derived from the CHelpG electrostatic potential fitting and subsequently adjusted to be consistent with the CHARMM27 charges and Lennard-Jones parameters were determined to reproduce interaction energies with water molecules. Bond, angle and torsion parameters were derived from the density-functional calculations and renormalized to maintain compatibility with the existing CHARMM27 parameters for standard residues. The extension of the CHARMM27 force field parameters for the non-standard biological residues presented here will have considerable use in simulations of ribozymes, including the study of freeze-trapped catalytic intermediates, metal ion binding and occupation, and thio effects.

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“…One explanation could be that the granule-associated RNAs could form secondary structure which makes them resistant to the RNase activities of eosinophil cationic protein and eosinophil-derived neurotoxin. A number of noncoding RNAs such as transfer RNA, ribosomal RNA and ribozymes contain sequences that can fold back on themselves to form a paired double helix and stem loops [35,36,37]. …”

Section: Discussionmentioning

confidence: 99%

Localization of DNA and RNA in Eosinophil Secretory Granules

Behzad

Walker

Abraham

et al. 2009

Int Arch Allergy Immunol

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Background: Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent ultrastructural evidence suggests that protein synthesis may also take place within eosinophilic granules. Methods: We used 2 different methods to detect the presence of DNA and RNA in eosinophil secretory granules. Using bromodeoxyuridine, a thymidine analogue, and bromouridine, a uracil analogue, we labeled the DNA and RNA in eosinophils in vivo in rabbits. Immunoelectron microscopy to localize these molecules was performed on ultrathin sections of blood and bone marrow eosinophils using monoclonal anti-bromodeoxyuridine antibody with IgG as a control. The immunogold grain density was measured in each subcellular compartment within the eosinophils and analyzed using image analysis software. A combination of DNA/CD63 immunofluorescence staining and a fluorescently labeled molecular probe that stains RNA was used to examine the presence of DNA and RNA in the secretory granules of human blood eosinophils. Results: The mean density of bromodeoxyuridine-labeled DNA and bromouridine-labeled RNA immunogold grains in the secretory granules of blood and bone marrow eosinophils were significantly higher (p < 0.0005) than cytoplasmic or background staining. We also demonstrated the existence of DNA and RNA in the CD63-positive secretory granules of human peripheral blood eosinophils by means of immunofluorescent staining and a fluorescently labeled molecular probe. Conclusions: These results provide evidence that eosinophil granules are the site of DNA and RNA synthesis and suggest the potential for a new role(s) for eosinophil-secretory granules.

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Ribozyme Structures and Mechanisms

Cited by 192 publications

References 143 publications

Quantum Mechanical/Molecular Mechanical Simulation Study of the Mechanism of Hairpin Ribozyme Catalysis

Quantum Mechanical/Molecular Mechanical Simulation Study of the Mechanism of Hairpin Ribozyme Catalysis

CHARMM force field parameters for simulation of reactive intermediates in native and thio‐substituted ribozymes

Localization of DNA and RNA in Eosinophil Secretory Granules

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