Mechanistic characterization of the HDV genomic ribozyme: Classifying the catalytic and structural metal ion sites within a multichannel reaction mechanism

Prior studies of the metal ion dependence of the self-cleavage reaction of the HDV genomic ribozyme led to a mechanistic framework in which the ribozyme can self-cleave by multiple Mg²⁺ ion-independent and -dependent channels [Nakano et al. (2001) Biochemistry 40, 12022]. In particular, channel 2 involves cleavage in the presence of a structural Mg²⁺ ion without participation of a catalytic divalent metal ion, while channel 3 involves both structural and catalytic Mg²⁺ ions. In the present study, experiments were performed to probe the nature of the various divalent ion sites and any specificity for Mg²⁺. A series of alkaline earth metal ions was tested for the ability to catalyze self-cleavage of the ribozyme under conditions that favor either channel 2 or channel 3. Under conditions that populate primarily channel 3, nearly identical K_ds were obtained for Mg²⁺, Ca²⁺, Ba²⁺, and Sr²⁺, with a slight discrimination against Ca²⁺. In contrast, under conditions that populate primarily channel 2, tighter binding was observed as ion size decreases. Moreover, [Co(NH₃)₆]³⁺ was found to be a strong competitive inhibitor of Mg²⁺ for channel 3 but not for channel 2. The thermal unfolding of the cleaved ribozyme was also examined, and two transitions were found. Urea-dependent studies gave m-values that allowed the lower temperature transition to be assigned to tertiary structure unfolding. The effects of high concentrations of Na⁺ on the melting temperature for RNA unfolding and the reaction rate revealed ion binding to the folded RNA, with significant competition of Na⁺ (Hill coefficient of ≈1.5-1.7) for a structural Mg²⁺ ion and an unusually high intrinsic affinity of the structural ion for the RNA. Taken together, these data support the existence of two different classes of metal ion sites on the ribozyme: a structural site that is inner sphere with a major electrostatic component and a preference for Mg²⁺, and a weak catalytic site that is outer sphere with little preference for a particular divalent ion.