C-terminal His-tagged aIF1 inhibits binding of tRNAi-Met and RNAo to ribosomes. (A) S. solfataricus ribosomes (0.1 μM) were incubated with RNAo (0.01 μM), a/eIF2·GTP· [35S]Met-tRNAi (lanes 2–4) and 0.3 μM CH-aIF1 (lane 3) or 0.3 μM NH-aIF1 (lane 4) at 70°C. (Lane 1) [35S]Met-tRNAi (1 μM) was incubated with ribosomes (0.1 μM). Samples were loaded on a native 4% polyacrylamide gel and the radioactive bands were visualized using a PhosphorImager (bottom). A graphical representation of two independent experiments is shown (top). The error bars represent standard deviations. (B) S. solfataricus ribosomes (0.1 μM) were incubated with a/eIF2·GTP·Met-tRNAi (lanes 2–4), the radioactively labeled RNAo (0.01 μM) and 1 μM CH-aIF1 (lane 3) or 1 μM NH-aIF1 (lane 4) at 70°C. (Lane 1) Only RNAo (0.01 μM) was incubated with ribosomes (0.1 μM). The samples were loaded on native 4% polyacrylamide gels and radioactive bands were visualized using a PhosphorImager (bottom). A graphical representation of two independent experiments is shown (top). The error bars represent standard deviations. (C) Both NH-aIF1 and CH-aIF1 bind to S. solfataricus ribosomes. S. solfataricus ribosomes (400 pmol) were incubated in the presence or the absence of either CH-aIF1 or NH-aIF1 and loaded on 0%–17% sucrose density gradients. The pellet fractions were resuspended in protein sample buffer and resolved on a 12% SDS-polyacrylamide gel using standard procedures. The proteins were blotted on nitrocellulose and the Western blots were probed for the presence of aIF1 as described above. (Lane 1) 50 pmol of purified CH-aIF1 was loaded on the gel as a positive control and marker for the Western blot. (Lanes 2,4) 200 pmol CH-aIF1 and 200 pmol NH-aIF1, respectively, were loaded on the gradients in the absence of ribosomes. (Lanes 3,5) 200 pmol CH-aIF1 and 200 pmol NH-aIF1, respectively, were loaded on the gradients upon incubation with 400 pmol of ribosomes.
