Elucidation of Catalytic Strategies of Small Nucleolytic Ribozymes from Comparative Analysis of Active Sites

Abstract

A number of small, self-cleaving ribozyme classes have been identified including the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), glmS, twister, hatchet, pistol, and twister sister ribozymes. Within the active sites of these ribozymes, myriad functional groups contribute to catalysis. There has been extensive structure-function analysis of individual ribozymes, but the extent to which catalytic devices are shared across different ribozyme classes is unclear. As such, emergent catalytic principles for ribozymes may await discovery. Identification of conserved catalytic devices can deepen our understanding of RNA catalysis specifically and of enzymic catalysis generally. To probe similarities and differences amongst ribozyme classes, active sites from more than 80 high-resolution crystal structures of self-cleaving ribozymes were compared computationally. We identify commonalities amongst ribozyme classes pertaining to four classic catalytic devices: deprotonation of the 2′OH nucleophile (γ), neutralization of the non-bridging oxygens of the scissile phosphate (β), neutralization of the O5′ leaving group (δ), and in-line nucleophilic attack (α). In addition, we uncover conservation of two catalytic devices, each of which centers on the activation of the 2′OH nucleophile by a guanine: one to acidify the 2′OH by hydrogen bond donation to it (γ′) and one to acidify the 2′OH by releasing it from nonproductive interactions by competitive hydrogen bonding (γ′′). Our findings reveal that the amidine functionalities of G, A, and C are especially important for these strategies, and help explain absence of U at ribozyme active sites. The identified γ′ and γ′′ catalytic strategies help unify the catalytic strategies shared amongst catalytic RNAs and may be important for large ribozymes, as well as protein enzymes that act on nucleic acids.

Cellular Small Molecules Contribute to Twister Ribozyme Catalysis

“…For instance, γ catalysis can be enhanced by acidifying the 2′-OH nucleophile and/or releasing the 2′-OH from inhibitory interactions. 4–6…”

Phosphoryl‐Transfer Reaction in RNA under Alkaline Conditions

“…The average crystallographic values of the O2′‐P‐O5′ angle for the four most frequent ribozymes are 140±27 and 76±16° for scissile and nonscissile phosphates, respectively . Nevertheless, the crystallographic values for the scissile phosphates can be questioned because the use of deoxyribonucleotide to prevent cleavage.…”

Recent Development of Glucosamine‐6‐phosphate Derivatives as Potential Antibacterial Agents

“…This high level of sequence conservation has been discovered in 5′‐UTR of the glmS mRNAs of B. subtilis and other 17 Gram‐positive bacteria . That is to say, in a large amount of Gram‐positive bacteria, the nucleotide sequences of the glmS riboswitch are quite similar and share the same metabolites and mechanisms of the self‐cleavage . This property makes it possible for the glmS riboswitch to serve as a broad‐spectrum antibiotic target.…”