Mg ²⁺ -Dependent Multistep Folding and Stabilization of the GAAA Tetraloop-Receptor Interaction in a Group I Intron

The GAAA tetraloop-receptor (TL-R) interaction is a ubiquitous tertiary interaction in RNA structures. Using the P4–P6 domain in the Tetrahymena thermophila group I intron as a model system, we studied the mechanism of TL-R formation using computer simulations. We show that the intron folds via a multistep pathway, populating seven states with distinct tertiary contacts. Under physiological Mg 2+ concentrations ([Mg 2+ ]), the loop-bulge-P4 tertiary interaction is essential to stabilize the docked TL-R complex, whereas in high [Mg 2+ ], the TL-R complex is stable by itself. The solvated Mg 2+ ions modulate the TL-R docking–undocking dynamics and stabilize non-native intermediate states. The condensation of Mg 2+ in the major grooves of the TL and R helices is critical for them to attain a specific stiffness essential for their facile docking. The results highlight the critical role of Mg 2+ ions in facilitating the formation of TL-R interactions that stabilize long-range tertiary contacts in the RNA structures.