Crucial Roles of Two Hydrated Mg²⁺ Ions in Reaction Catalysis of the Pistol Ribozyme

Abstract

Pistol ribozymes constitute anew class of small selfcleaving RNAs.Crystal structures have been solved, providing three-dimensional snapshots along the reaction coordinate of pistol phosphodiester cleavage,c orresponding to the precatalytic state,avanadate mimic of the transition state,a nd the product. The results led to the proposed underlying chemical mechanism. Importantly,ahydrated Mg 2+ ion remains innersphere-coordinated to N7 of G33 in all three states,a nd is consistent with its likely role as acid in general acid base catalysis (d and b catalysis). Strikingly,t he new structures shed light on as econd hydrated Mg 2+ ion that approaches the scissile phosphate from its binding site in the pre-cleavage state to reacho ut for water-mediated hydrogen bonding in the cyclophosphate product. The major role of the second Mg 2+ ion appears to be the stabilization of product conformation. This study delivers amechanistic understanding of ribozyme-catalyzed backbone cleavage.

Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes

“…165,[202][203][204] Later, this view was expanded because intensive atomic mutagenesis-based assays shed light on the mechanistic role of divalent metal ions. 205,206 The first three-dimensional structures of the pistol ribozyme were available in 2016, solved by X-ray crystallographic methods with a resolution of 2.7 Å (env25 pistol) 203 and 3.0 Å (env27 pistol), 204 respectively. Both structures represent a precatalytic conformation that has been trapped based on mutants carrying a hydrogen atom instead of the native 2 0 OH nucleophile at the cleavage site.…”

“…8B) and the product (2 0 ,3 0 cyclophosphate). 206 This broad structural foundation resulted in a profound proposal of the underlying chemical mechanism. Essentially, a hydrated Mg 2+ ion remains innersphere-coordinated to the N7 nitrogen of G33 in all three states, which supports its proposed role as acid in general acid base chemistry (d and b catalysis).…”

“…Essentially, a hydrated Mg 2+ ion remains innersphere-coordinated to the N7 nitrogen of G33 in all three states, which supports its proposed role as acid in general acid base chemistry (d and b catalysis). 206 Interestingly, a second hydrated Mg 2+ ion approaches the to-be-cleaved phosphate from its binding site in the pre-cleavage state to stretch out for water-mediated hydrogen bonding to one of the non bridging oxygen atoms of the cyclophosphate product. 206 The major role of the second Mg 2+ ion appears to be stabilization of the product conformation.…”

Impact of 3-deazapurine nucleobases on RNA properties

“…Suitable deazanucleosides for informative RNA atomic mutagenesis experiments are 7-deazaadenosine (c 7 A) ( 4 , 11–13 , 15 , 16 ), 3-deazaadenosine (c 3 A) ( 14 , 20 ), 1-deazaadenosine (c 1 A) ( 11 , 12 , 14 , 20 ), 7-deazaguanosine (c 7 G) ( 13 , 15 ) and 3-deazacytidine (c 3 C) ( 13 , 21 ). Moreover, 3-deazaguanosine (c 3 G) and 1-deazaguanosine (c 1 G) would be highly useful for RNA atomic mutagenesis, however, such studies are very rare ( 22 ) because synthetic access to appropriate phosphoramidite building blocks and to the corresponding RNAs is challenging ( 22 , 23 ).…”

Changed reactivity of secondary hydroxy groups in C8-modified adenosine – lessons learned from silylation

“…Oligoribonucleotides carrying site-specific modifications are highly required as models for structure and function studies, driven by the ongoing discovery of new RNAs and their investigation [1][2][3][4][5][6]. This has put demand also on synthetic chemistry to provide suitable compounds at monomeric and oligomeric level.…”