Understanding Catalysis of Phosphate‐Transfer Reactions by the Large Ribozymes

Abstract

Large ribozymes are unique among catalytic RNA molecules in that their reactions involve intermolecular nucleophilic attack on an RNA phosphodiester linkage. Crystal structures of near-atomic resolution are now available for the group I and group II self-splicing introns and the RNA subunit of RNase P. The structural data agrees well with the earlier models proposed on the basis of biochemical studies and the evidence at hand suggests that all of the large ribozymes utilize a mechanism in which coordination of Mg(II) ions reduces the negative charge on the scissile phosphodiester linkage, as well as assists both the nucleophilic attack and the departure of the leaving group.

Mercury(II)-Catalyzed Cleavage, Isomerization and Depurination of RNA and DNA Model Compounds and Desulfurization of Their Phosphoromonothioate Analogs

“…The ratio of [1a] to [1b] approached unity, corresponding to essentially equal values for k 2 and k −2 . With cleavage, the contributions of pH-independent and hydroxide ion catalyzed reactions to the observed pseudo first-order rate constant may be expressed by Equation (2).…”

“…Metal ion-catalyzed cleavage of both RNA and DNA phosphodiester linkages has been investigated extensively to understand the mechanism of action of natural nucleases (including ribozymes) [1][2][3][4][5], as well as to develop artificial ones [6][7][8][9][10]. Comparative studies with phosphodiester and phosphorothioate model compounds have been instrumental in revealing coordination of catalytically important metal ions to the scissile linkage [11][12][13][14][15][16].…”

Phosphodiester models for cleavage of nucleic acids

“…The underlying idea behind the latter application is that protonation of the leaving group by a general acid is not needed with 5´-thiosubstituted analogs, since the sulfide ion is a much better leaving group than the alkoxide ion. Most extensively used thiosubstitution, however, is replacement of either one of the non-bridging oxygens with sulfur, which allows stereochemical studies based on the so-called rescue effect [ 41 – 42 ]. When non-bridging oxygen that participates in binding of Mg 2+ is replaced with sulfur, the activity drops, but may be restored by using a soft Lewis acid, such as Mn 2+ or Zn 2+ .…”

“…Transesterification reactions catalyzed by the large ribozymes (group I and II introns, the lariat capping ribozyme, the spliceosome and RNAse P) share a common mechanism that sets them apart from reactions catalyzed by small ribozymes or protein enzymes [ 42 , 105 ]. Perhaps most strikingly, the large ribozymes do not make use of the vicinal 2´-OH as a nucleophile but instead fold into an elaborate tertiary structure that allows an external nucleophile to attack the phosphorus atom of the scissile phosphodiester linkage [ 106 – 107 ].…”

Impact of steric constraints on the product distribution of phosphate-branched oligonucleotide models of the large ribozymes

“…While the reaction conditions used in this study are too alkaline for hybridization of the oligonucleotides, the ribozymes work under physiological conditions and recognition of the reactive site by Watson-Crick base-pairing seems to be a common strategy. 13,14, 22 In other words, in the reactions catalysed by the large ribozymes the reactive phosphodiester linkage is typically embedded in a double-helical stem which, in all likelihood, has an effect on the product distribution that is similar to but greater than the one reported in this paper. Accordingly, the observed regioselectivity of the large ribozymes may largely be explained by steric constraints imposed by the highly ordered environment of the catalytic centre.…”