Journal ArticleDOI
Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom.
TLDR
It is confirmed that thio substitution has no effect on the nonenzymatic alkaline cleavage of RNA and the strong preference of Mg2+ for binding to oxygen rather than sulfur suggest that the pro-RP oxygen atom is not coordinated to Mg 2+ in the E.S complex or in the transition state.Abstract:
The L-21 ScaI ribozyme derived from the intervening sequence of Tetrahymena thermophila pre-rRNA catalyzes a guanosine-dependent endonuclease reaction that is analogous to the first step in self-splicing of this intervening sequence. We now describe pre-steady-state kinetic experiments, with sulfur substituting for the pro-RP (nonbridging) phosphoryl oxygen atom at the site of cleavage, that test aspects of a kinetic model proposed for the ribozyme reaction (Herschlag, D., & Cech, T. R. (1990) Biochemistry 29, 10159-10171). Thio substitution does not affect the reaction with subsaturating oligonucleotide substrate and saturating guanosine ((kcat/Km)S), consistent with the previous finding that binding of the oligonucleotide substrate limits this rate constant. In contrast, there is a significant decrease in the rate of single-turnover reactions of ribozyme-bound (i.e., saturating) oligonucleotide substrate upon thio substitution, with decreases of 2.3-fold for the reaction with guanosine ((kcat/Km)G) and 7-fold for hydrolysis [i.e., with solvent replacing guanosine; kc(-G)]. These "thio effects" are consistent with rate-limiting chemistry, as shown by comparison with model reactions. Nonenzymatic nucleophilic substitution reactions of the phosphate diester, methyl 2,4-dinitrophenyl phosphate monoanion, are slowed 4-11-fold by thio substitution for reactions with hydroxide ion, formate ion, fluoride ion, pyridine, and nicotinamide. In addition, we have confirmed that thio substitution has no effect on the nonenzymatic alkaline cleavage of RNA (Burgers, P. M. J., & Eckstein, F. (1979) Biochemistry 18, 592-596). Considering the strong preference of Mg2+ for binding to oxygen rather than sulfur, the modest thio effect on the chemical step of the ribozyme-catalyzed reaction and the absence of a thio effect on the equilibrium constant for binding of the oligonucleotide substrate suggest that the pro-RP oxygen atom is not coordinated to Mg2+ in the E.S complex or in the transition state. General implications of thio effects in enzymatic reactions of phosphate diesters are discussed.read more
Citations
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Journal ArticleDOI
MODIFIED OLIGONUCLEOTIDES: Synthesis and Strategy for Users
Sandeep Verma,Fritz Eckstein +1 more
TL;DR: The range, scope, and practical utility of chemically modified oligonucleotides that contain backbone, base, and sugar modifications to investigate the mechanism and stereochemical aspects of biochemical reactions are reviewed.
Journal ArticleDOI
Kinetics and Mechanisms for the Cleavage and Isomerization of the Phosphodiester Bonds of RNA by Brønsted Acids and Bases
Journal ArticleDOI
Metal ion catalysis in the Tetrahymena ribozyme reaction
TL;DR: It is concluded that the Tetrahymena ribozyme is a metalloenzyme, with mechanistic similarities to several protein enzymes9–12, and contributes directly to catalysis by coordination to the 3′ oxygen atom in the transition state, presumably stabilizing the developing negative charge on the leaving group.
Journal ArticleDOI
Structure and Mechanism of DNA Polymerase β
TL;DR: In this review, recent crystallographic studies of pol β in various liganded and conformational states during the insertion of right and wrong nucleotides as well as during the bypass of damaged DNA are described.
Journal ArticleDOI
Role of divalent metal ions in the hammerhead RNA cleavage reaction.
Sue Ann C Dahm,Olke C. Uhlenbeck +1 more
TL;DR: Results suggest certain divalent ions either participate directly in the cleavage mechanism or are specifically involved in stabilizing the tertiary structure of the hammerhead.
References
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Journal ArticleDOI
Enzyme-Catalyzed Phosphoryl Transfer Reactions
TL;DR: This paper presents a meta-analyses of six classes of enzyme involving reactions at phosphorus and some of the mechanisms leading to these reactions are described.
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Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant.
TL;DR: These studies provide the first complete and self-consistent thermodynamic descriptions of DNA polymerase and establish the basis for quantitative assessment of the reactions contributing to its extraordinary fidelity.
Journal ArticleDOI
The chemistry of self-splicing RNA and RNA enzymes.
TL;DR: The chemical mechanisms of RNA-catalyzed reactions are discussed with emphasis on the self-splicing ribosomal RNA precursor of Tetrahymena and the enzymatic activities of its intervening sequence RNA.
Journal ArticleDOI
Conserved sequences and structures of group I introns: building an active site for RNA catalysis — a review
TL;DR: Sequence elements conserved among the available set of 66 group I introns are compiled and the possible significance of conserved nucleotides within base-paired duplexes is discussed.