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

Simple Summary The class I ligase is an in-vitro-evolved ribozyme with a high catalytic turnover. In the present study, we considered the conditions under which this ribozyme retains ligation activity by removing the partial structure and by splitting. The ligation activity was maintained even when the structure was split into two molecules of 55 and 39 nucleotides. Our study clarified in several cases the length of the duplexes that is necessary to facilitate activity of the class I ligase ribozyme assembled from multiple fragments. 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 similar to 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 similar to 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.