General Acid–Base Catalysis Mediated by Nucleobases in the Hairpin Ribozyme

Abstract

The catalytic mechanism by which the hairpin ribozyme accelerates cleavage or ligation of the phosphodiester backbone of RNA has been incompletely understood. There is experimental evidence for an important role for an adenine (A38) and a guanine (G8), and it has been proposed that these act in general acid-base catalysis. In this work we show that a large reduction in cleavage rate on substitution of A38 by purine (A38P) can be reversed by replacement of the 5′-oxygen atom at the scissile phosphate by sulfur (5′-PS), which is a much better leaving group. This is consistent with A38 acting as the general acid in the unmodified ribozyme. The rate of cleavage of the 5′-PS substrate by the A38P ribozyme increases with pH log-linearly, indicative of a requirement for a deprotonated base with a relatively high pKa. On substitution of G8 by diaminopurine, the 5′-PS substrate cleavage rate at first increases with pH and then remains at a plateau, exhibiting an apparent pKa consistent with this nucleotide acting in general base catalysis. Alternative explanations for the pH dependence of hairpin ribozyme reactivity are discussed, from which we conclude that general acid-base catalysis by A38 and G8 is the simplest and most probable explanation consistent with all the experimental data.

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

“…Purine bases are most prevalent, and the majority of ribozymes use a deprotonated guanine nucleobase as a general base in the cleavage reaction (16,19,(39)(40)(41)(42)(43)(44)(45).…”

“…protonated and unprotonated respectively) and thus the rate of reaction is strongly pH dependent. This leads to a classic bell-shaped dependence of reaction rate on pH (46), and the adenine and guanine nucleotides were shown to be the general acid and base respectively in the cleavage reaction by phosphorothiolate substitution (45,47). The twister ribozyme also uses an adenine plus guanine combination, but with two significant differences as discussed below.…”

Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM

“…It is now generally accepted that ribozymes like the HDVr use a variety of catalytic strategies, including site-specific shifts of nucleobase pK a 's and/or recruitment of divalent metal ions to stabilize electrostatically strained structures (Fedor 2009;Schnabl and Sigel 2010;Wong and Pollack 2010;Golden et al 2013;Ward et al 2014;Panteva et al 2015). These motifs are well established from detailed experimental and theoretical analysis of the hairpin and hammerhead ribozymes (Lee et al 2007(Lee et al , 2009Leclerc 2010;Suydam et al 2010;Cottrell et al 2011;Kath-Schorr et al 2012).…”

Crystal structure of Pistol, a class of self-cleaving ribozyme

“…Mutation of the nucleobase involved in general acidbase catalysis in other self-cleaving ribozymes causes significantly reduced activity. For example, in HDV, VS, and Hairpin ribozymes, nucleobase mutation on the proposed general acid severely reduces their activity (21)(22)(23). Additional experiments, such as the substitution of the bridging O5′ atom of U11 to a 5′-phosphorothiolate, which serves as a better leaving group than oxygen and does not require protonation from the general acid, will be helpful to clarify the role of A32U and A32C mutations in Pistol catalysis (21)(22)(23)(24).…”