Imidazole Rescue of a Cytosine Mutation in a Self-Cleaving Ribozyme

Abstract

Ribozymes use a number of the same catalytic strategies as protein enzymes. However, general base catalysis by a ribozyme has not been demonstrated. In the hepatitis delta virus antigenomic ribozyme, imidazole buffer rescued activity of a mutant with a cytosine-76 (C76) to uracil substitution. In addition, a C76 to adenine substitution reduced the apparent pKa (where Ka is the acid constant) of the self-cleavage reaction by an amount consistent with differences in the pKa values of these two side chains. These results suggest that, in the wild-type ribozyme, C76 acts as a general base. This finding has implications for potential catalytic functions of conserved cytosines and adenines in other ribozymes and in ribonuclear proteins with enzymatic activity.

How RNA acts as a nuclease: some mechanistic comparisons in the nucleolytic ribozymes

“…Structural and mechanistic evidence indicates that the HDV ribozyme uses metal ion-bound water as the general base, and a cytosine nucleobase as a general acid (15,(64)(65)(66)(67). The TS ribozyme also appears to employ a hydrated metal ion as the general base, but at the present time we have not identified the general acid with confidence.…”

HDV Family of Self-Cleaving Ribozymes

“…2,65,80 The CPEB3 sequence also exhibits a flat pH profile around pH 7, as is observed in both HDV ribozymes under similar conditions. 2,45 Sequence-based searches using the human CPEB3 sequence revealed a remarkable conservation of this motif across mammalian CPEB3 genes (Fig. 4.5).…”

“…Mutating the genomic C75 or antigenomic C76 residue to a U or G results in a loss of catalytic activity. 39,40,[44][45][46] The J4/2 strand is further stabilized by the A78 residue of the genomic ribozyme via an A-minor tertiary interaction with the C18 and G29 residues that form the middle base pair of the P3 helix. [34][35][36]47 A purine nucleotide is required 5 0 to A78 to allow for hydrogen bonding between its WatsonCrick face and the 2 0 OH of the C19 nucleotide at the base of P3.…”

“…Nathan Riccitelli and Andrej Lupták respectively, can be summarized as follows: (1) in all crystal structures, the nucleobase is located within several Å ngstroms of the scissile phosphate [34][35][36] ; (2) the C75/76U and C75/76G variants do not support catalysis and C75/ 76A shows only partial activity 45,46 ; (3) the kinetic pH profile of the C75/ 76A variant shows an apparent pK a shifted by a number of pH units corresponding to the difference between the pK a values of free cytidine and adenosine 46 ; (4) substitution of the active-site cytosine with cytosine analogs similarly shifts the apparent kinetic pK a by approximately the same amount as the pK a difference between the free bases 67,68 ; (5) self-scission of the inactive C75/76U or deleted C75/76 at this position can be rescued by exogenous nitrogenous bases including free cytosine, imidazole, and other analogs with the kinetic pK a values tracking the pK a values of the free bases 45,69,70 ; and (6) proton inventory experiments and solvent kinetic isotope effects of the C75, C75A, and imidazole-rescued inactive ribozymes support a proton transfer from this position in the ribozyme (Fig. 4.3).…”

RNA catalysis: ribozymes, ribosomes, and riboswitches

“…14 The inactive C75U mutation could be rescued with imidazole and the pK a of the reaction appeared to track with the pK a of nucleobases substituted at the key residue. 15,16 This suggested that C75 might function as a general acid to protonate the O5′ leaving group. However, subsequent crystal structures of the substrate form of the ribozyme showed that the C75 N3 is closer to the nucleophile, instead implicating C75 as a general base to deprotonate the 2′-OH.…”