Hatchet ribozyme structure and implications for cleavage mechanism

Significance Self-cleaving ribozymes are RNAs that catalyze position-specific cleavage of their phosphodiester backbone. The cleavage site of the newly discovered hatchet ribozyme is located at the very 5′ end of its consensus secondary structure motif. Here we report on the 2.1-Å crystal structure of the hatchet ribozyme in the product state, which defines its intricate tertiary fold and identifies key residues lining the catalytic pocket. This in turn has allowed us to propose a model of the precatalytic state structure and a role in catalysis for a conserved guanine. This study therefore provides a structure-based platform toward an improved understanding of the catalytic mechanism of hatchet ribozymes. Small self-cleaving ribozymes catalyze site-specific cleavage of their own phosphodiester backbone with implications for viral genome replication, pre-mRNA processing, and alternative splicing. We report on the 2.1-Å crystal structure of the hatchet ribozyme product, which adopts a compact pseudosymmetric dimeric scaffold, with each monomer stabilized by long-range interactions involving highly conserved nucleotides brought into close proximity of the scissile phosphate. Strikingly, the catalytic pocket contains a cavity capable of accommodating both the modeled scissile phosphate and its flanking 5′ nucleoside. The resulting modeled precatalytic conformation incorporates a splayed-apart alignment at the scissile phosphate, thereby providing structure-based insights into the in-line cleavage mechanism. We identify a guanine lining the catalytic pocket positioned to contribute to cleavage chemistry. The functional relevance of structure-based insights into hatchet ribozyme catalysis is strongly supported by cleavage assays monitoring the impact of selected nucleobase and atom-specific mutations on ribozyme activity.