Scientific Report 2005

Molecular Biology

Mechanisms of RNA Assembly and Catalysis

M.J. Fedor, E.M. Calderon, J.W. Cottrell, C.P. Da Costa, J.W. Harger, Y.I. Kuzmin, E.M. Mahen

Recent evidence that RNA catalysis participates in regulation of gene expression as well as in RNA processing and protein synthesis underscores the importance of learning the molecular basis of ribozyme activity. The hairpin ribozyme is an especially good model for investigating RNA catalytic mechanisms because of its relative simplicity and the availability of high-resolution structures that provide a framework for evaluating structure-function relationships. This ribozyme catalyzes reversible phosphodiester cleavage through attack of a ribose 2´ oxygen nucleophile on an adjacent phosphorus (Fig. 1). Our goals have been to identify which parts of the ribozyme contribute to catalysis and to understand the chemical basis of this activity.

Fig. 1. Chemical mechanism of RNA cleavage mediated by the family of small catalytic RNAs that includes the hairpin ribozyme. Cleavage proceeds through an SN2-type mechanism that involves in-line attack of the 2´ oxygen nucleophile on the adjacent phosphorus to form a trigonal bipyramidal transition state in which 5 electronegative oxygen atoms form transient bonds with phosphorus. Breaking of the 5´ oxygen-phosphorus bond generates products with 5´ hydroxyl and 2´,3´-cyclic phosphate termini.

Like all enzymes, hairpin ribozymes combine several strategies to enhance catalytic rate. One important strategy, which is apparent from crystal structures, is the alignment of nucleophilic and leaving-group oxygens in the optimal orientation for an SN₂-type nucleophilic attack. Biochemical and structural studies also implicate 2 active-site nucleobases, guanine 8 and adenine 38, in catalytic chemistry; the N-1 ring nitrogen of guanine 8 is located near the 2´ oxygen that acts as the nucleophile during cleavage, and the N-1 ring nitrogen of adenine 38 is located near the 5´ oxygen leaving group.

Ribonuclease A is a protein enzyme that catalyzes the same chemical reaction as hairpin ribozyme cleavage and has 2 active-site histidines that occupy positions similar to those of guanine 8 and adenine 38. Ribonuclease A provides a textbook example of concerted general acid-base catalysis, and the similarity between hairpin ribozyme and ribonuclease A active-site structures led to the idea that guanine 8 and adenine 38 might serve as general acid and base catalysts as the histidines of ribonuclease A do. The activity of the hairpin ribozyme increases with increasing pH, consistent with the notion that activity depends on the availability of guanine 8, in its unprotonated form, to accept a proton to activate the 2´ hydroxyl nucleophile as proposed in the general acid-base catalysis model. However, a ribozyme variant in which guanine 8 is replaced by an abasic residue has the same pH dependence as an unmodified ribozyme, suggesting that the pH transition in activity does not involve guanine 8. These data support an alternative model in which the protonated form of guanine 8 donates hydrogen bonds that provide electrostatic stabilization as negative charge develops in the transition state (Fig. 2). Replacing adenine 38 with an abasic residue, on the other hand, does eliminate this pH-dependent transition, evidence that the protonation state of adenine 38 is important for activity.

Fig. 2. Results of mechanistic studies of the hairpin ribozyme are consistent with 2 models in which the functional form of adenine 38 is either protonated or unprotonated. In the first model (A), protonated adenine 38 would act as a general acid by donating a proton to the 5´ oxygen, acting in concert with hydroxide ion that activates the 2´ oxygen nucleophile during cleavage, and unprotonated adenine 38 would act as a general base to activate the 5´ oxygen nucleophile during ligation. In the second model (B), unprotonated adenine 38 accepts a hydrogen bond from the 5´ hydroxyl nucleophile during ligation and accepts a hydrogen bond from a protonated bridging 5´ oxygen during cleavage, providing electrostatic stabilization to developing negative charge. In both models, the amidine group of guanine 8, in its protonated form, donates hydrogen bonds to the 2´ and phosphoryl oxygens that stabilize the negative charge that develops in the transition state and that position reactive groups in the orientation appropriate for an SN2 in-line nucleophilic attack.

The activity that is lost when adenine 38 or guanine 8 is replaced by abasic residues can be rescued by certain nucleobases provided in solution. The molecules that can rescue activity all have planar structures and an amidine group, that is, an amino group in α-position to a ring nitrogen. The same feature is shared with the Watson-Crick face of the missing adenine and guanine, suggesting that chemical rescue occurs through binding of exogenous nucleobases in the cavity left by an abasic substitution. Purines that lack an amidine group can inhibit chemical rescue, presumably by competing with rescuing nucleobases for binding in the cavity left by the abasic substitution. Thus, rescue does not occur through binding alone, and amidine functional groups must form specific stabilizing interactions with the transition state. The pH dependence of chemical rescue of ribozymes lacking adenine 38 changes according to the intrinsic basicity of the rescuing nucleobase. These and other results are consistent with 2 models of the hairpin ribozyme catalytic mechanism in which adenine 38 contributes either general acid-base catalysis (Fig. 2A) or electrostatic stabilization of negative charge that develops as 5 electronegative oxygen atoms form transient bonds with phosphorus in the transition state (Fig. 2B).

Publications

Fedor, M.J., Williamson, J.R. The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6:399, 2005.

Kuzmin, Y.I, Da Costa, C.P., Cottrell, J.W., Fedor, M.J. Role of an active site adenine in hairpin ribozyme catalysis. J. Mol. Biol. 349:989, 2005.

Mahen, E.M., Harger, J.W., Calderon, E.M., Fedor, M.J. Kinetics and thermodynamics make different contributions to RNA folding in vitro and in yeast. Mol. Cell 19:27, 2005.