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 i...