Figure 3.

Schematic description of the method for determining the affinities of rescuing metal ions. (A) Equilibrium for binding of Mn²⁺ (red) to metal site A in the free ribozyme (K^Mn), and reaction of S_3′S and S_3′O with Mg²⁺ and Mn²⁺ bound at site A, with relative rate constants of k_Mg^rel and k_Mn^rel, respectively (k^rel = k^S_3′S/k^S_3′O). The ribozyme active site is schematically represented by the thin outline, with the IGS of the ribozyme shown by shaded letters. Addition of Mn²⁺ would lead to partial occupancy of all metal ion sites, depending on the metal ion concentration and the Mn²⁺ affinity of each site. This is represented by the Mn²⁺ occupancy of metal sites B and C in the ^Mn⋅E and [^Mn⋅E_⋅G^⋅S]^‡ species. (B) The Mn²⁺ concentration dependence of the observed reactivity of S_3′S relative to S_3′O (k_obsd^rel) predicted from the model of A. Analysis of the Mn²⁺ concentration dependence gives the Mn²⁺ affinity for metal site A in the free ribozyme, as described in the text.