Scientific Report 2006

Molecular Biology

Mechanisms of RNA Assembly and Catalysis

M.J. Fedor, E.M. Calderon, J.W. Cottrell, C.P. Da Costa, S. Daudenarde, J.W. Harger, Y.I. Kuzmin, E.M. Mahen, M. Roychowdhury-Saha

Our goal is to generate basic insights into catalysis by RNA and RNA-protein enzymes, RNA folding, and RNA interactions with small molecules. In addition to contributing basic knowledge of RNA structure and function in normal growth and development, results of our studies provide a framework for developing technical and therapeutic applications involving RNAs as targets and reagents.

Apart from the ribosome, which catalyzes peptidyl transfer, the naturally occurring ribozymes catalyze transfer of phosphate groups. The small RNA enzymes that we study catalyze reversible phosphodiester cleavage reactions that generate 5′ hydroxyl and 2′,3′-cyclic phosphate termini (Fig. 1). Possible strategies for catalysis of phosphoryl transfer reactions include aligning reactive groups in an optimal orientation for an in-line attack mechanism, general acid-base catalysis of proton transfer to activate nucleophilic oxygens or to stabilize oxyanion-leaving groups, electrostatic stabilization of negative charge that accumulates in the transition state, and destabilizing the ground state. Our goal is to understand which of these catalytic strategies RNA enzymes use.

>Fig. 1. Chemical mechanism of RNA cleavage mediated by the family of small catalytic RNAs that includes the hairpin ribozyme. Cleavage of the phosphodiester bond occurs 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. Breaking of the 5′ oxygen-phosphorus bond generates products with 5′ hydroxyl and 2′,3′-cyclic phosphate termini.

In contrast to the chemical versatility of the amino acid side chains that make up the active sites of protein enzymes, just 4 nucleotides are available for the construction of ribozyme active sites. Nucleotides are well suited to faithful storage and transmission of genetic information through complementary base pairing, but they are not particularly adept at catalytic chemistry. Protonation and deprotonation of nucleotides occur at high or low pH extremes, a situation that would make it difficult to mediate general acid or base catalysis at neutral pH. No positively charged nucleotide functional groups are expected to be available at neutral pH to function as Lewis acids to activate a nucleophile or stabilize an electronegative transition state or an oxyanion-leaving group.

Recent high-resolution structures of self-cleaving RNAs lay the groundwork for experiments to probe fundamental questions about how RNA enzymes use their functional groups for catalysis. Like all enzymes, hairpin ribozymes combine several strategies to enhance catalytic rate. One important strategy, apparent from the crystal structures, is the alignment of nucleophilic and leaving-group oxygens in the optimal orientation for an in-line S_N2-type nucleophilic attack. The structure of the hairpin ribozyme active site places guanine 8, adenine 9, adenine 10, and adenine 38 nucleobases near the reactive phosphate. Guanine 8 and adenine 38 occupy positions reminiscent of 2 histidine residues in the active site of ribonuclease A, a protein enzyme that catalyzes the same reaction. Histidine residues perform general acid-base catalysis during ribonuclease A catalysis, so the similarity between hairpin ribozyme and ribonuclease A active sites raised the possibility that guanine 8 and adenine 38 nucleobases might perform functions similar to those of histidine residues.

Hairpin ribozyme activity increases with increasing pH, consistent with the notion that activity depends on the availability of the deprotonated form of guanine 8 to accept a proton from the 2′ hydroxyl nucleophile as predicted by the general acid-base catalysis model. To test this model, we replaced guanine 8 with an abasic residue, a substitution that eliminates the nucleobase but leaves the phosphodiester backbone intact. However, this abasic variant had the same pH dependence as an unmodified ribozyme, arguing that the pH transition does not involve guanine 8. Replacing adenine 38 with an abasic residue, on the other hand, did eliminate the pH-dependent transition in activity, implicating adenine 38 in a catalytically important deprotonation.

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 in the transition state as 5 electronegative oxygen atoms from transient bonds with phosphorus (Fig. 2B) and guanine 8 donates hydrogen bonds to stabilize the transition state electrostatically.

Fig. 2. Two models of hairpin ribozyme catalysis. 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 negative charge that develops in the transition state and positions reactive groups in the orientation appropriate for an SN2 in-line nucleophilic attack. Reproduced with permission from Fedor, M.J., Williamson, J.R. The catalytic diversity of RNAs. Nat. Rev. Mol. Cell Biol. 6:399, 2005. Copyright 2005 Nature Publishing Group/Macmillan Magazines Ltd.