Linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of the Tetrahymena ribozyme

We have explored the linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of Tetrahymena thermophila ribozyme by examining the Mg²⁺-induced folding and the urea-induced denaturation of the folded state as a function of Na⁺ under equilibrium folding conditions using hydroxyl radical footprinting. These studies allowed a thermodynamic examination of eight discrete protection sites within P4-P6 that are involved in several tertiary structure contacts. Monovalent ions compete with Mg²⁺ ions in mediating P4-P6 folding. The urea denaturation isotherms demonstrated ΔΔG values of > 2 kcal mol^-1 in experiments conducted in 10 versus 200 mM NaCl at a constant 10 mM MgCl₂. However, the individual-site isotherms reported by footprinting revealed that larger than average changes in ΔG values were localized to specific sites within the Mg²⁺-rich A-bulge. The competitive effects of monovalent ions were less when K⁺ rather than Na⁺ was the monovalent cation present. This result indicates the importance of the specific K⁺ binding sites that are associated with AA-platform structures to P4-P6 folding and stability. These site-specific footprinting data provide quantitative and site-specific measurements of the ion-linked stability for P4-P6 that are interpreted with respect to crystallographic data.