Probing General Base Catalysis in the Hammerhead Ribozyme

Abstract

Recent structural and computational studies have shed new light on the catalytic mechanism and active site structure of the RNA cleaving hammerhead ribozyme. Consequently, specific ribozyme functional groups have been hypothesized to be directly involved in general/acid base catalysis. In order to test this hypothesis, we have developed an affinity label to identify the functional general base in the S. mansoni hammerhead ribozyme. The ribozyme was reacted with a substrate analogue bearing a 2'-bromoacetamide group in place of the nucleophilic 2'-hydroxyl group which would normally be deprotonated by a general base. The electrophilic 2'-bromoacetamide group is poised to alkylate the general base, which is subsequently identified by footprinting analysis. Herein, we demonstrate alkylation of N1 of G12 in the hammerhead ribozyme in a pH and [Mg(2+)] dependent manner that is consistent with the native cleavage reaction. These results provide substantial evidence that deprotonated N1 of G12 functions directly as a general base in the hammerhead ribozyme; moreover, our experiments provide evidence that the pKa of G12 is perturbed downward in the context of the active site structure. We also observed other pH-independent alkylations, which do not appear to reflect the catalytic mechanism, but offer further insight into ribozyme conformation and structure.

Role of tautomerism in RNA biochemistry

“…-dependent manner that is consistent with the direct involvement of the N1 of G12 in the cleavage reaction (Thomas and Perrin 2008).…”

“…5C;McKay 1996;Martick and Scott 2006;Thomas and Perrin 2008). The N1 of the catalytic guanosine is within hydrogen bonding distance (3.5 Å) from the 2 ′ -OH nucleophile (Martick and Scott 2006).…”

The chemical versatility of RNA

“…Mutation of G8 reduces catalysis 10 4 -fold, but this effect is rescued if the bridging oxygen of the scissile phosphate is replaced with a sulphur leaving group, alleviating the need for a general acid [13]. In another study, a bromoacetamide group was attached to the 2 0 -oxygen nucleophile, which would produce a covalent adduct if a general base were in the vicinity [14]. Indeed, G12 becomes covalently linked to the nucleophile in this case.…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…421 A number of other ribozymes have been examined: a crystal structure of the hammerhead ribozyme in the presence of Mn(II) ions shows a network of hydrogen bonding around the metal ion that facilitates proton transfer during the cleavage reaction. 422 General base catalysis in the hammerhead ribozyme using the bromoacetamide derivative of 2 0 -aminocytidine has been reported, 423 the use of fluorinated purine derivatives in nucleotide analogue interference mapping has been demonstrated using the Varkud satellite ribozyme, 224 and the identification of a manganese-dependent ribozyme found in the 3 0 -UTR of Xenopus Vg1 mRNA. 424 The role of C75 as a general acid in the cleavage of the hepatitis delta virus has been studied using a double mutant ribozyme, and it was concluded that it does not require divalent metal ions in its role as acid.…”