Cleavage Reaction

The mechanism of the backbone cleavage reaction was first inferred from the products (Wu et al. 1989) (Fig. 2a). A 5' hydroxyl group and 2',3'-cyclic phosphate group on the cleavage products are consistent with a mechanism in which the oxygen at the 2' position adjacent to the scissile phosphate attacks the phosphorus and the phosphorus to 5'-oxygen bond is broken. This reaction is common to the small self-cleaving ribozymes, but it is distinct from the reaction catalyzed by the large ribozymes where a 3' hydroxyl group is generated (RNase P RNA, group I and group II intron-derived ribozymes). For the small ribozymes, it is hypothesized that the 5' hydroxyl group displacement reaction occurs with inversion of configuration through a pentavalent transition state where the attacking 2' oxygen and leaving 5' oxygen are at the apical positions of a trigonal bipyramidal arrangement of the phosphorane

Fig.2a-c Cleavage reaction and possible catalytic mechanisms for the HDV ribozymes. a Backbone cleavage results in a 2',3'-cyclic phosphate and a 5'hydroxyl group. Proposed catalytic groups include abase (B:) to facilitate deprotonation of the 2' hydroxyl group (the nucleophile) and an acid (B:H+) to donate a proton to the 5' bridging oxygen (the leaving group). Potential catalytic groups, as discussed in the text, include a Mg2+ ion-bound water (Mg2+HO:H or Mg+HO:) and an active site nucleobase (H+:Cyt75/76 or :Cyt75/76). Two transition states (TSi and TS2) are shown but in a concerted mechanism, concurrent proton transfer might occur. b :Cyt75/76 acting at the 2'hydroxyl group (a general base in the cleavage reaction). c H+:Cyt75/76 acting at the 5' bridging oxygen (a general acid in the cleavage reaction)

oxygens. Inversion of configuration about the phosphorus, when a phospho-rothioate stereoisomer is placed at the cleavage site, is strong support for this mechanism in the hammerhead and hairpin ribozymes (van Tol et al. 1990; Slim and Gait 1991); however, similar studies have not been reported for the HDV ribozymes.

The reverse reaction, attack by the 5' hydroxyl on the cyclic phosphate center to restore the 3',5' linkage, has yet to be demonstrated with the HDV ribozymes. The lack of a detectable ligation reaction seems surprising given the chemistry of the reaction (Gerlt et al. 1975), the ability of both the hammerhead and hairpin ribozymes to catalyze ligation (Feldstein and Bruening 1993; Hegg and Fedor 1995; Hertel and Uhlenbeck 1995), and the biology in which RNA replication would appear to benefit from ribozyme-catalyzed ligation. Nevertheless, two reports of in vitro ligation in the HDV ribozyme system have not been born out. The idea that a conformational change in the RNA may help drive the forward reaction is consistent with results from biophysical (Harris et al. 2002; Pereira et al. 2002) and structural studies (Ke et al. 2004) that reveal structural differences in the ribozyme domain between the precursor and product forms. In addition, a reaction that favors cleavage over ligation is consistent with the absence of binding interactions for both the 5' fragment and a catalytic metal ion in the 3' product following cleavage (Shih and Been 2001a; Ke et al. 2004).

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