Abstract
Self-aminoacylating ribozymes catalyze the attachment of amino acids to RNA, serving as pivotal models to investigate the catalytic roles of RNA in prebiotic evolution. In this study, we investigated how divalent metal ions (Mg(2+) and Ca(2+)) modulate local and global structures in two such ribozymes, S-1A.1-a and S-2.1-a, using 4-cyanotryptophan (4CNW) fluorescence and native gel electrophoresis. By tracking 4CNW fluorescence changes at varying concentrations of Mg(2+) and Ca(2+) and temperatures, we determined how these ions influence the catalytic sites and overall conformations of the ribozymes. Our findings reveal that Mg(2+) specifically binds to S-1A.1-a at low concentrations, stabilizing the local structure around the aminoacylation site and causing the site to become more buried, which is essential for catalytic activity. Although higher Mg(2+) and Ca(2+) concentrations induce global structural rearrangements, these shifts have minimal impact on the local environment of the aminoacylation site, underscoring the dominance of local structural stability in sustaining ribozyme function. In contrast, the activity of S-2.1-a effectively adapts to both Mg(2+) and Ca(2+), and its fluorescence results indicate a more solvent-exposed aminoacylation site. Overall, these data highlight that local structural changes in the ribozyme's catalytic core are more critical for its function than global conformational shifts. Our study highlights the importance of local environmental changes in ion-dependent ribozyme catalysis and provides insights into the molecular mechanisms of self-aminoacylating ribozymes.