Figure 5.

Effect of the addition of 39S subunits on the mRNA-independent binding of [³⁵S]fMet-tRNA formed in the presence and absence of IF3_mt. (A) The 28S subunits were incubated with [³⁵S]fMet-tRNA and IF2_mt in the absence (closed circles) or presence (open circles) of IF3_mt. Reaction mixtures were analyzed by sucrose density gradient centrifugation and the position of the [³⁵S]fMet-tRNA was located by filtering appropriate fractions as described in Materials and Methods. (B) [³⁵S]fMet-tRNA binding was initially carried out with 28S subunits in the presence of IF2_mt but in the absence of mRNA. Reactions mixtures were prepared in the absence (closed circles) or presence (open circles) of IF3_mt. Following assembly of these complexes, 39S subunits (0.066 µM) were added and the incubation was continued for an additional 5 min at 27°C. The resulting complexes were then analyzed on sucrose gradients.