Catalytic RNA or ribozymes present excellent systems to study the foundational principles of biological catalysis. In addition, they serve as models for investigating RNA structure and its relation to function. We report the first crystal structures of the Varkud satellite (VS) ribozyme, the largest among endonucleolytic ribozymes. The structures reveal a modular organization, in which independently folding domains are assembled into the functional conformation of the ribozyme by three-way junctions. The catalytic domain of the ribozyme recognizes and binds the substrate through tertiary interactions, and substrate docking is accompanied by remodeling of substrate structure that results in the formation of a catalytically-relevant active site. The catalytic strategies employed by the VS bear resemblance to that used by protein and DNAzyme ribonucleases, highlighting the robustness of the chemistry of catalytic RNA cleavage. In addition, the active site of the VS ribozyme is strikingly similar to that of the hairpin and hammerhead ribozymes, although the three endonucleolytic ribozymes have distinct sequences and structures. The presence of these architectural features in the context of what appears to be distinct mechanisms of catalysis underscores their functional importance and bolsters the case for convergent evolution. However, our understanding of the possible mechanisms for the emergence of distinct endonucleolytic ribozyme function during evolution and the ease of access to distinct catalytic motifs involved in RNA cleavage is limited. We have explored the mutational connections between the VS, hairpin and hammerhead ribozymes and delineated plausible pathways by which these distinct ribonuclease motifs can be accessed via intersection of their neutral networks. Intersections between neutral networks are possible due to the existence of bifunctional sequences that exhibit catalytic functions corresponding to both networks. We have identified two such bifunctional sequences that can support hairpin and VS, and hammerhead and hairpin dual functions. Bifunctional sequences present plausible evolutionary nodes toward increasing complexity in functional RNA as illustrated by the hammerhead, hairpin and VS ribozymes in our study. Our results provide a framework to investigate the evolutionary origins of distinct catalytic function in RNA.




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