Molecular Anatomy of the Class I Ligase Ribozyme for Elucidation of the Activity-Generating Unit

The class I ligase ribozyme consists of 121 nucleotides and shows a high catalytic rate comparable to that found in natural proteinaceous polymerases. In this study, we aimed to identify the smaller active unit of the class I ligase ribozyme comprising ~50 nucleotides, comparable to the estimated length of prebiotically synthesized RNA. Based on the three-dimensional structure of the class I ligase ribozyme, mutants were prepared and their ligation activities were analyzed. Sufficient ligation activity was maintained even when shortening to 94 nucleotides. However, because it would be difficult to approach the target of ~50 nucleotides by removing only the partial structure, the class I ligase ribozyme was then split into two molecules. The ligation activity was maintained even when splitting into two molecules of 55 and 39 nucleotides. Using a system with similar split ribozymes, we analyzed the ligation activity of mutants C30, C47, and A71, which have been previously identified as the positions that contribute to catalytic activity, and discussed the structural basis of the activity of these bases. Our findings suggest the rationale for the class I ligase ribozyme's assembling from multiple fragments that would be achievable with prebiotic synthesis.