FIGURE 3.

Reconstitution of Stem I from interaction of the Specifier and Common half molecules. (A) Formation of the Specifier/Common complex was observed by polyacrylamide gel electrophoresis (PAGE) mobility shift analyses. Specifier 2AP₉₈ half molecule (183 μM) was titrated with increasing concentrations of the Common half molecule (lane 2, 0.0; 3, 23.9; 4, 47.9; 5, 71.8; 6, 95.8; 7, 120; 8, 144; 9, 287 μM). RNA standards, a 17-mer anticodon stem and loop and unfractionated tRNA ∼76 mer were run in lanes 1 and 10. In addition, lane 1 contained Specifier half molecule (25 mer), and lane 10 contained Common half molecule (27 mer). (B) Complex formation between the Common half molecule and the two Specifier half molecules, 2AP₉₈ (black square) and 2AP₁₀₂ (red circle). Ethidium bromide stained RNAs of the PAGE mobility shift analyses were quantified with ImageQuant; the results were normalized and plotted as percent Complex (bound) vs. concentration of the Common half molecule. Binding curves were analyzed with a nonlinear regresssion (Origin) to extrapolate dissociation constants. The error for multiple determinations of the K _ds was determined to be ±1.0–±2.3 μM.