Mg²⁺-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 Mg²⁺ concentrations ([Mg²⁺]), the loop-bulge-P4 tertiary interaction is essential to stabilize the docked TL-R complex, whereas in high [Mg²⁺], the TL-R complex is stable by itself. The solvated Mg²⁺ ions modulate the TL-R docking-undocking dynamics and stabilize non-native intermediate states. The condensation of Mg²⁺ 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 Mg²⁺ ions in facilitating the formation of TL-R interactions that stabilize long-range tertiary contacts in the RNA structures.