News and Publications

Mechanisms of RNA Assembly and Catalysis

The ways that RNA enzymes can accomplish catalysis are of considerable interest, particularly because of the recent finding that RNA catalysis is responsible for protein synthesis. Our focus is the hairpin ribozyme, a small RNA enzyme that catalyzes a reversible phosphodiester cleavage (Fig. 1). Progress in understanding this simple ribozyme will help illuminate the types of catalytic strategies available to RNA enzymes.

Like proteins, RNAs can form compact, ordered structures and can bind macromolecules and small ligands with high affinity and specificity. Clearly, both RNA and protein enzymes can promote catalysis through positioning and orientation of reactive groups. However, the ribonucleosides that make up RNA enzymes lack the chemical versatility of the amino acids that make up protein enzymes. Until the hairpin ribozyme was proved to be an exception, many researchers thought that all RNA enzymes required metal cations for catalysis. Our early studies indicated that cations support assembly of the functional ribozyme structure but do not participate directly in catalytic chemistry. The ability of the ribozyme to mediate catalysis without metal cations points to a direct role for RNA functional groups in catalytic chemistry.

Several detailed views of the active site of the hairpin ribozyme recently became available when F. D'Amaré and colleagues solved crystal structures for hairpin ribozymes complexed with products and with substrate and transition-state analogs. For the first time, it became possible to identify the functional groups close to the reactive phosphodiester.

We developed an experimental approach in which exogenous nucleobase rescue of abasic ribozymes is used to probe potential catalytic roles of specific RNA functional groups. For this approach, we prepared ribozyme variants that lack specific active-site nucleobases. The loss of active-site residues interferes with activity, but in some instances, activity is restored when certain nucleobase analogs are provided in solution. Comparison of the nucleobases that do or do not rescue activity allows us to identify structural and biochemical features important for catalysis.

Deletion of one active-site guanine residue, G8, reduced activity by more than 350-fold. Cytosine, isocytosine, 2,6-diaminopurine, and 2-aminopyridine, but no other nucleobase analogs, restored cleavage activity when provided in solution. Each of these nucleobase analogs has the same guanidinium group as the nucleobase that normally occupies this position. These structural similarities suggest that rescuing molecules compensate for the missing guanine by binding in the cavity left by its deletion.

Unexpectedly, we found that rescue activity increased sharply with decreasing pH, suggesting that rescue requires protonation. This result supports a novel electrostatic stabilization model for RNA catalysis in which a cationic nucleobase neutralizes the negative charge that develops in the transition state as 5 electronegative oxygens transiently bond with phosphorus (Fig. 2A). Alternatively, interaction with a cationic nucleobase might activate the 2´ oxyanion nucleophile, or the nucleobase could act as a general acid to protonate the leaving group during the breaking of 5´ oxygen-phosphorus bonds (Fig. 2B).

We examined the reverse reaction of ligation to distinguish between these models. A cationic nucleobase should contribute electrostatic stabilization to the transition state for both cleavage and ligation reactions, because the transition state is identical in forward and reverse reactions. In contrast, a nucleobase that contributes general acid catalysis to the cleavage reaction should contribute general base catalysis to ligation as the 5´ oxygen becomes the nucleophile and the 2´ oxygen becomes the leaving group. If exogenous cytosine provides general base catalysis, ligation activity would reflect the abundance of cytosine in the deprotonated state and ligation rescue activity would increase with increasing pH. Instead, rescue by exogenous cytosine has the same pH-rate profile for ligation as for cleavage. These results provide strong support for the novel electrostatic stabilization model.

Publications

Fedor, M.J. The catalytic mechanism of the hairpin ribozyme. Biochem. Soc. Trans. 30(Pt. 6):1109, 2002.

Fedor, M.J. Determination of kinetic parameters for hammerhead and hairpin ribozymes. Methods Mol. Biol., in press.

Fedor, M.J. RNA biochemistry from A to Z. Cell 109:20, 2002.

Fedor, M.J. The role of metal ions in RNA catalysis. Curr. Opin. Struct. Biol. 12:289, 2002.

Fedor, M.J., Westhof, E. Ribozymes: the first 20 years. Mol. Cell 10:703, 2002.