The Conventional Hairpin Ribozyme (HP-WT)

The conventional hairpin ribozyme is derived from the minus strand of Tobacco ringspot virus satellite RNA [12a,b]. It works in cis (intramolecularly) in nature, but

corrected transcript

Fig. 5.2.3. Schematic representation of RNA cis and trans splicing by group I introns.

it has been engineered to work in trans (intermolecularly) and thus catalyze site-specific reversible cleavage at the 5' side of a GUC triplet (Figure 5.2.5), generating a 2',3'-cyclic phosphate and a free 5'-hydroxyl group (Figure 5.2.6) [13].

The ribozyme consists of two independently folded domains, A and B. Each of these contains an internal loop (A, B) flanked by two Watson-Crick helices (H-1, H-2 in domain A, H-3, H-4 in domain B). The RNA substrate is bound to the ri-bozyme to generate Helix 1 and 2 and is reversibly cleaved within loop A. Catalysis is supported by positively charged metal ions, typically Mg2+. On the basis of several biochemical studies it has been suggested the metal ions are important for

Fig. 5.2.4. Strategy for the repair of genetic disorders by use of twin ribozymes.
Fig. 5.2.5. Secondary structure of the conventional hairpin ribozyme HP-WT. The arrow denotes the cleavage site. The four helices (H-1 through H-4) are marked by bars.
Fig. 5.2.6. Mechanism of the reversible cleavage of a phosphodiester bond catalyzed by the hairpin ribozyme.

stabilization of the ribozyme active conformation and not as thought for a long time, to participate in active site chemistry. Last, the crystal structure, which has recently been solved [14], showed that no metal ions are present in the active center, providing additional proof for the structural role of positively charged co-factors.

Folding into the active conformation requires a sharp bend between helix 2 and helix 3 to orient the two helical domains in an antiparallel fashion and to enable Loop A to interact with Loop B (Figure 5.2.7). Catalysis is achieved by specific positioning of the phosphodiester bond to be processed within the ribozyme architecture, such that nucleophilic in line attack of the 2 '-OH at the phosphorus atom in an SN2-like mechanism becomes possible (Figure 5.2.6).

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