Figure 1.

Magnesium-dependent folding of the Tetrahymena pre-RNA. (A) ³²P-labeled pre-RNA was annealed at 95°C or incubated at 30°C in buffer containing the concentration of MgCl₂ indicated above each lane, before loading on a native 6% polyacrylamide gel at 10°C (see Materials and Methods). Band 1, folding intermediate (I); band 4, RNA that is competent to self-splice (N) (29). Diffuse radioactivity above and below band 1 is defined as unfolded RNA (U). (B) Mg²⁺ dependence of I and N after annealing at 95°C. ●, fraction of native pre-RNA (ƒ_N); ○, fraction of intermediate (ƒ_I). The fraction of unfolded RNA (ƒ_U) is obtained from ƒ_U + ƒ_I + ƒ_N = 1. For clarity, ƒ_U is not plotted. ƒ_N and ƒ_I were fit to the Hill equation independently as described in Materials and Methods. Values were C_mα = 4.1 ± 0.03 and 4.2 ± 0.05 mM, for N and I respectively, and n = 9.0 ± 0.8. C, Fraction of I and N after 2 hr at 30°C in splicing buffer plus MgCl₂ and 10% glycerol. Symbols and curves are as in B. C_mN = 3.4 ± 0.01; C_mI = 3.5 ± 0.02 mM; n_N = 15 ± 0.7 and n_I = 17 ± 1. (D) Derivative of ƒ_N with respect to Mg²⁺ concentration. Values of C_max and (df_x/dC)_max were obtained analytically from the curve in B. ΔC is the width of the curve at ½ (dƒ_α/dC)_max.