Files
Abstract
The hovlinc ribozyme is a recently discovered self-cleaving ribozyme in human ncRNA. We determined the structure of the active ribozyme to 4.1 Å using cryo-EM and the inactive ribozyme to 3.05 Å using X-ray crystallography. Both of these methods utilized a synthetic anti-RNA antibody fragment, Fab BL3-6 and its antigenic hairpin as a fiduciary module to aid in data collection and analysis. Both of these structures depict a global topology with two coaxial stacks of helices and a double pseudoknot. The active site of the cryo-EM model showed the five residues organized in two layers: a base triple and a noncanonical purine-purine interaction. Using extensive atomic mutations and combinations of multiple mutations to introduce and then suppress perturbations in cleavage activity, I validated the catalytic relevance of these active site interactions predicted by the cryo-EM model. From this mutational analysis, we predict a Mg2+ ion interacts with a non-bridging oxygen of the scissile phosphate to aid in catalysis. A guanosine is involved in splaying the scissile phosphate for nucleophilic attack and is positioned so that its N1 can act as a general base catalyst. This active site bears a striking resemblance to that of the crystal structure of the pistol ribozyme. I applied the experiments that probed the active site interactions in the hovlinc ribozyme to the similar interactions in the pistol ribozyme. The results validated the purine-purine juxtaposition and base pair in the base triple shown in the pistol ribozyme crystal structure and are more consistent with the crystal structure of the active site than proposed rearrangements of the active site. I could not get direct evidence for the other base triple interactions in the pistol ribozyme, because of mechanistic and contextual differences between the pistol and hovlinc ribozymes. This work is a significant step forward in understanding human self-cleaving ribozymes and demonstrates valuable strategies for studying ribozyme active site structures.