Figure 4.

Effect of E.coli IF3, IF3_mt and its derivatives on the binding of [³⁵S]fMet-tRNA to mitochondrial 28S subunits in the presence and absence of mRNA. Initiation complexes were prepared containing [³⁵S]fMet-tRNA, IF2_mt and 28S subunits in the presence and absence of poly(A,U,G) as mRNA. After incubation with the indicated amounts of IF3_mt or its derivatives, the amount of complex remaining was determined using a nitrocellulose filter-binding assay. The data are reported as the relative amount of binding obtained since data from different experiments were combined and the absolute numbers obtained depend on the percentage of active 28S subunits in different preparations. For these experiments, the value normalized to 1 for binding in the presence of mRNA represents 0.71 pmol while the value bound in the absence of mRNA was 0.35 pmol. For the experiments testing the effects of deletion of the N- and C-terminal extension, the level of binding obtained in the absence of mRNA and IF3_mt was 0.12 pmol. IF3_mtFL in the presence of mRNA (closed circles), IF3_mtFL in the absence of mRNA (closed squares), IF3_mtΔN (open circles) or IF3_mtΔC (open triangles) or E.coli IF3 (open squares) were added as indicated. The amount of fMet-tRNA retained on the small subunit was measured as described in Materials and Methods.