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

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 among ribozyme classes, active sites from more than 80 high-resolution crystal structures of self-cleaving ribozymes were compared computationally. We identify commonalities among ribozyme classes pertaining to four classic catalytic devices: deprotonation of the 2'OH nucleophile (gamma), neutralization of the nonbridging oxygens of the scissile phosphate (beta), neutralization of the O5' leaving group (delta), and in-line nucleophilic attack (alpha). 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 (gamma') and one to acidify the 2'OH by releasing it from nonproductive interactions by competitive hydrogen bonding (gamma ''). Our findings reveal that the amidine functionalities of G, A, and C are especially important for these strategies and help explain the absence of U at ribozyme active sites. The identified gamma' and gamma '' catalytic strategies help unify the catalytic strategies shared among catalytic RNAs and may be important for large ribozymes, as well as protein enzymes that act on nucleic acids.