Nucleobase Participation in Ribozyme Catalysis

Zhong, Zhao; McLeod, Aileen; Harusawa, Shinya; Araki, Lisa; Yamaguchi, Mitsugu; Kurihara, Takushi; Lilley, David M.J.

doi:10.1021/ja0502775

Cited by 56 publications

(44 citation statements)

References 19 publications

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“…First, the ring nitrogen atoms of the imidazole are 2-4 Å from where N1 or N3 of guanine would be placed assuming that the ribose is not moved; participation of the imidazole in the reaction is therefore likely to require transient distortion of the active site. Similarly slow rates of cleavage were observed in the corresponding A756Im-idazole VS ribozyme (Zhao et al 2005) and in HDV C76U in the presence of exogenous imidazole (Perrotta et al 1999). Second, the modified ribozyme will have lost some transitionstate stabilization in the absence of hydrogen bonding to G8.…”

Section: Resultsmentioning

confidence: 69%

“…8A). A discrete product of the correct size was generated in significant amounts over this timescale; the ultimate yield will be limited by the hydrolysis of the cyclic phosphate, which becomes important at longer times (Zhao et al 2005). The observed reaction rate was k obs = 0.024 min ÿ1 .…”

Section: Resultsmentioning

confidence: 99%

“…We have recently developed a novel chemo-genetic strategy for investigating the role of nucleobases in ribozyme chemistry by means of the site-specific replacement of a conventional nucleotide base by imidazole (Harusawa et al 1996;Araki et al 2004). We demonstrated catalytic activity in a VS ribozyme, in which a critical adenosine was replaced by imidazole (Zhao et al 2005). We have now explored the role of the nucleobase of G8 in hairpin ribozyme catalysis using the imidazole nucleoside.…”

Section: Introductionmentioning

confidence: 99%

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Nucleobase catalysis in the hairpin ribozyme

Wilson

Ouellet²,

Zhong³

et al. 2006

RNA

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RNA catalysis is important in the processing and translation of RNA molecules, yet the mechanisms of catalysis are still unclear in most cases. We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed between guanine and adenine bases. We show that a modified ribozyme in which guanine 8 has been substituted by an imidazole base is active in both cleavage and ligation, with ligation rates 10-fold faster than cleavage. The rates of both reactions exhibit bell-shaped dependence on pH, with pK a values of 5.7 6 0.1 and 7.7 6 0.1 for cleavage and 6.1 6 0.3 and 6.9 6 0.3 for ligation. The data provide good evidence for general acid-base catalysis by the nucleobases.

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Section: Resultsmentioning

confidence: 69%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Nucleobase catalysis in the hairpin ribozyme

Wilson

Ouellet²,

Zhong³

et al. 2006

RNA

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“…Instead, these reactions can be investigated by using kinetic solvent isotope experiments in which the reaction rate is measured by using RNA and reaction solutions that have been reconstituted with D 2 The data in Fig. 1 A (open circles) show that, like the reaction in H 2 O, the cleavage rate of RG RNA in D 2 O exhibited a bell-shaped dependence on pD (Ϫlog 10 [deuterium ion]) and also fit well to the model in Fig. 1C.…”

Section: Resultsmentioning

confidence: 99%

“…For example, Strobel and colleagues (9) observed pH-dependent rescue of a ligation reaction by using nucleotide analog substitutions at position 756 in the putative active-site loop of the ribozyme but not at other positions. Also, Lilley and coworkers (10) showed that substitution of A756 with imidazole supported cleavage and ligation, although it was not reported whether this effect was unique to position 756 or whether the reaction was affected by pH.…”

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confidence: 99%

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme

Smith

Collins²

2007

Proc. Natl. Acad. Sci. U.S.A.

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A fast-cleaving version of the Varkud satellite ribozyme, called RG, shows an apparent cis-cleavage rate constant of 5 sec ؊1 , similar to the rates of protein enzymes that catalyze similar reactions. Here, we describe mutational, pH-rate, and kinetic solvent isotope experiments that investigate the identity and rate constant of the rate-limiting step in this reaction. Self-cleavage of RG exhibits a bell-shaped rate vs. pH profile with apparent pKas of 5.8 and 8.3, consistent with the protonation state of two nucleotides being important for the rate of cleavage. Cleavage experiments in heavy water (D2O) revealed a kinetic solvent isotope effect consistent with proton transfer in the rate-limiting step. A mutant RNA that disrupts a peripheral loop-loop interaction involved in RNA folding exhibits pH-and D2O-independent cleavage Ϸ10 3 -fold slower than wild type, suggesting that this mutant is limited by a different step than wild type. Substitution of adenosine 756 in the putative active-site loop with cytosine also decreases the cleavage rate Ϸ10 3 -fold, but the A756C mutant retains pH-and D2O-sensitivity similar to wild type, consistent with this mutant and wild type being limited by the chemical step of the reaction. These results suggest that the RG ribozyme provides a good experimental system to investigate the nature of fast, rate-limiting steps in a ribozyme cleavage reaction.kinetic solvent isotope effect ͉ kinetics ͉ Neurospora ͉ pH vs. rate S ince the discovery of RNA catalysis, several natural RNAs and many more RNA and DNA sequences obtained by in vitro selection have been shown to catalyze the cleavage and/or ligation of phosphodiester bonds, as well as other chemical activities. Recent work has begun to investigate the range of catalytic mechanisms used by ribozymes and to understand the similarities and differences with protein enzymes (1-3). The local environment of a folded RNA can shift the pK a of certain nucleobases by two or more pH units into the range where they could function as proton donors or acceptors in general acidbase catalysis, similar to histidines in their protein counterparts (4). Charged nucleobases could also participate in electrostatic stabilization in the transition state. Indeed, the bell-shaped rate vs. pH curves typical of protein enzymes that use general acid-base catalysis have been observed for certain hepatitis delta virus (HDV) ribozymes (5, 6) and Varkud satellite (VS) ribozyme (this study).Most previously characterized versions of the Neurospora VS ribozyme showed rather slow cis-or trans-cleavage apparent rate constants (k obs ) in the range of 1 min Ϫ1 or less and were only slightly affected by pH between pH 5.5 and 9.0 (7); however, a trans-ligating construct did exhibit pH dependence below pH 7.0 (8). Other observations have also raised the possibility that a protonated group could be involved in the rate-limiting step of a VS ribozyme reaction pathway. For example, Strobel and colleagues (9) observed pH-dependent rescue of a ligation reaction by using nucleo...

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Mass Determination of Phosphoramidites

Harusawa

Fujitake

Kurihara

et al. 2006

CP Nucleic Acid Chemistry

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Nucleoside phosphoramidites are the most widely used building blocks in contemporary solid-phase synthesis of oligonucleotides. The accurate molecular weight measurements of such molecules, which are acid-labile compounds, may be easily determined by mass spectrometry using a matrix system, triethanolamine/NaCl, on a liquid secondary ion mass spectrometer (LSIMS) or fast-atom bombardment (FAB) MS equipped with a double-focusing mass spectrometer. The present method rapidly and easily measures the accurate molecular weights of various phosphoramidites as adduct ions [M+Na]+ with an average mass error smaller than 0.4 ppm, allowing determination of the formulas of the phosphoramidites in place of elemental analysis. Further, it was found that intensities of molecular-related ions could be enhanced to the highest degree by adjustment of the molar ratio of phosphoramidite and NaCl, fixing the amount of triethanolamine on LSIMS, making the present method a powerful tool for identification of phosphoramidites by mass spectrometry.

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Nucleobase Participation in Ribozyme Catalysis

Cited by 56 publications

References 19 publications

Nucleobase catalysis in the hairpin ribozyme

Nucleobase catalysis in the hairpin ribozyme

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme

Mass Determination of Phosphoramidites

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