Nucleobase-mediated general acid-base catalysis in the Varkud satellite ribozyme

Abstract

Existing evidence suggests that the Varkud satellite (VS) ribozyme accelerates the cleavage of a specific phosphodiester bond using general acid-base catalysis. The key functionalities are the nucleobases of adenine 756 in helix VI of the ribozyme, and guanine 638 in the substrate stem loop. This results in a bell-shaped dependence of reaction rate on pH, corresponding to groups with pK a ¼ 5.2 and 8.4. However, it is not possible from those data to determine which nucleobase is the acid, and which the base. We have therefore made substrates in which the 5′ oxygen of the scissile phosphate is replaced by sulfur. This labilizes the leaving group, removing the requirement for general acid catalysis. This substitution restores full activity to the highly impaired A756G ribozyme, consistent with general acid catalysis by A756 in the unmodified ribozyme. The pH dependence of the cleavage of the phosphorothiolatemodified substrates is consistent with general base catalysis by nucleobase at position 638. We conclude that cleavage of the substrate by the VS ribozyme is catalyzed by deprotonation of the 2′-O nucleophile by G638 and protonation of the 5′-O leaving group by A756. 5′-phosphorothiolate | RNA catalysis | nucleolytic ribozymes | catalytic mechanism R ibozyme-mediated catalysis is important for both RNA splicing and translation (1), yet its chemical origins are incompletely understood. The nucleolytic ribozymes bring about the site-specific cleavage or ligation of RNA, with an acceleration of a millionfold or greater. The intensively studied protein RNase A catalyses an identical cleavage reaction, and much evidence supports the hypothesis that each of these phosphoryl transfer reactions is subject to general acid-base catalysis. This mechanism requires a general base to deprotonate the attacking nucleophile, and a general acid to protonate the oxyanion leaving group (Fig. 1).The most common chemical entities implicated in RNA catalysis by the nucleolytic ribozymes are the nucleobases (2). Guanine appears to play a catalytic role in the hairpin, hammerhead, GlmS, and Varkud satellite (VS) ribozymes, adenine in the hairpin, and VS and cytosine in the hepatitis delta virus (HDV) ribozyme. Crystal structures of the hairpin ribozyme (3) reveal the presence of guanine (G8) and adenine (A38) bases juxtaposed with the 2′-O and 5′-O, respectively, of the scissile phosphate, where they seem poised to act in general acid-base catalysis. This is consistent with the pH dependence of the reaction (4) and its variation with functional group modifications (5-8).In its simplest active form, the VS ribozyme comprises five helices (II through VI) organized by two three-way junctions, which acts in trans upon a substrate stem loop (helix I) with an internal loop that contains the scissile phosphate (Fig. 1). The loop also contains the critical G638 (9). A756 (10-13) is contained within an internal loop in helix VI. While no crystal structure of the VS ribozyme has yet been solved, a small-angle X-ray scattering-derived model plac...

Do the hairpin and VS ribozymes share a common catalytic mechanism based on general acid–base catalysis? A critical assessment of available experimental data

“…The pH profile of cleavage rate for the G638DAP plus 59-PO substrate was bell-shaped, corresponding to pK a values of 4.8 and 5.6 (Wilson et al 2007), consistent with general acid-base catalysis by A756 and DAP at position 638. In contrast, with the 59-PS substitution the reaction rate increased to pH z6 and remained at a plateau at higher pH (Wilson et al 2010). A constant rate is to be expected at higher pH once the base is fully deprotonated since deprotonation of the acid is no longer relevant.…”

“…Substitutions in the ribozyme that impair the function of the general acid, lowering the activity of the oxy substrate, should have little effect on cleavage of a 59-PS-containing substrate. We therefore made a 59-phosphorothiolate substitution at the scissile phosphate of the VS ribozyme (Wilson et al 2010). We found that the cleavage activity of VS A756G was impaired 1000-fold on the oxy (59-PO) substrate, but the activity was completely restored for the 59-PS-containing substrate.…”

“…Both are formed through the interaction of two internal loops. In both cases, an active guanine lies on the opposing strand of the internal loop harboring the scissile phosphate, while an active adenine is provided by the second loop (Rupert and Ferré-D'Amaré 2001;Wilson et al 2010). In the crystal structure of the hairpin ribozyme the positions of G8 and A38 are consistent with roles of base and acid, respectively, in the cleavage reaction, corresponding to the proposed functions of G638 and A756 in the VS ribozyme.…”

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

“…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.…”

Mechanistic insights and functionalization of alkynes in homogeneous gold catalysis.

“…General base catalysis is a common occurrence in biological systems where strong bases are not tolerated and where deprotonation or proton transfer is mediated by weak bases through hydrogen-bonding interaction. [128][129][130] In gold catalysis, the role of a hydrogen bond acceptor (S in Scheme 11) is more complex than that of a general base. Because S is aurophilic to some extent, it competes with an alkyne/alkene starting material in their complexation with cationic gold (Scheme 11).…”