Dissection of the ion-induced folding of the hammerhead ribozyme using ¹⁹F NMR

NOTE: THE MATHEMATICAL SYMBOLS IN THIS ABSTRACT CANNOT BE DISPLAYED CORRECTLY ON THIS PAGE. PLEASE REFER TO THE PUBLISHER'S WEBSITE FOR AN ACCURATE DISPLAY. We have used ¹?F NMR to analyze the metal ion-induced folding of the hammerhead ribozyme by selective incorporation of 5- fluorouridine.Wehave studied the chemical shift and linewidths of ¹?F resonances of 5-fluorouridine at the 4 and 7 positions in the ribozyme core as a function of added Mg²?. The data fit well to a simple two-state model whereby the formation of domain 1 is induced by the noncooperative binding of Mg²? with an association constant in the range of 100 to 500 M?¹, depending on the concentration of monovalent ions present. The results are in excellent agreement with data reporting on changes in the global shape of the ribozyme. However, the NMR experiments exploit reporters located in the center of the RNA sections undergoing the folding transitions, thereby allowing the assignment of specific nucleotides to the separate stages. The results define the folding pathway at high resolution and provide a time scale for the first transition in the millisecond range.