FIGURE 6.

The levels of the three initiation factors IF1, IF2, and IF3 increase after cold shock and determine an imbalance of the IF/ribosome stoichiometric ratio. S30 extracts prepared from control (A) and cold-shocked (120 min) (B) cells were fractionated by centrifugation on 10%–30% sucrose density gradients. The amount of each initiation factor present in the resulting gradient fractions (left to right from bottom to top of gradient) were determined following SDS-PAGE, Western blotting, and immunoquantification as described in Materials and Methods. An analysis similar to that shown in panels A and B was also carried out on S30 extracts derived from cells cold-shocked for 30, 60, and 90 min. (C) The total levels (i.e., the sum of the values of each gradient fraction) of IF1 (black), IF2 (dark gray), and IF3 (light gray), normalized with respect to the amount of ribosomes in each extract, as a function of the time of cold shock. The data in C do not contain error bars, as the quantification yielded only one value for each initiation factor at each indicated time of cold shock. However, similar experiments were carried out three times for IF1 and four times for IF2 and IF3, producing essentially the same results, namely, after 2-h cold shock at 10°C, the relative increases with respect to the pre-cold-shock levels were the same as those shown in C (±20% for IF3, ±25% for IF1 and IF2).