FIGURE 7.

Effect of YafO on the stability of translation initiation complex. A, effect of YafO on the formation of the ompA mRNA-70 S Ribosome-tRNA_f^Met complex. Lane 1, without YafO-His₆; lane 2, 1 μm tRNA_f^Met and 0.05 μm 70 S ribosomes; lanes 3–11, 1 μm tRNA_f^Met and 0.05 μm 70 S ribosomes and 5.43, 2.72, 1.36, 0.68, 0.34, 0.17, 0.085, 0.0425, and 0.021 μm YafO-His₆, respectively. B, primer extension analysis of the ompA mRNA after phenol extraction. The experiment was carried out in the same way as described for Fig. 7A except that reaction products in lanes 4 and 6 were phenol-extracted to remove proteins before primer extension. Lane 1, without YafO-His₆; lane 2, 1.36 μm YafO-His₆; lane 3, 1 μm tRNA_f^Met and 0.05 μm 70 S ribosomes; lane 4, 1 μm tRNA_f^Met and 0.05 μm 70 S ribosomes; lane 5, 0.05 μm 70 S ribosomes, 1 μm tRNA_f^Met with 1.36 μm YafO-His₆; lane 6, 0.05 μm 70 S ribosomes, 1 μm tRNA_f^Met with 1.36 μm YafO-His₆. C, binding of [³⁵S]methionine-tRNA_f^Met to 70 S ribosomes at 37 °C in the absence and the presence of different amounts of YafO-His_6. 70 S ribosomes (0.05 μm) were first incubated with [³⁵S]methionine-tRNA_f^Met (1 μm) and the ompA mRNA (0.035 μm) for 10 min at 37 °C then 0, 0.021, 0.0425, 0.085, 0.34, 0.68, 1.36, 2.72, or 5.43 μm YafO was added. The reaction mixtures were incubated for additional 10 min at 37 °C and then applied to nitrocellulose filters (Millpore; 0.45 μm hemagglutinin), which were washed twice with 2 ml of buffer A before measuring the radioactivity.