Figure 1

Tunable repression of eEF3 expression leads to a gradual decrease in growth rate. (a) The P_MET25-YEF3 (VKY8) strain was grown at 30 °C in liquid synthetic complete medium lacking methionine and cysteine (SC-met-cys) supplemented with methionine at different concentrations (see insert). The growth rate (µ₂) was calculated as the slope of the linear regression of log₂-transformed OD₆₀₀ measurements. (b) Wild type (VKY9) and P_MET25-YEF3 (VKY8) strains were grown overnight in SC-met-cys medium, 10-fold serially diluted, spotted on SC-met-cys plates supplemented with the indicated concentration of methionine, and incubated at 30 °C for two days. (c) Western blot analysis of the P_MET25-YEF3 strain grown in SC-met-cys medium supplemented with indicated methionine concentrations. In addition to eEF3, the blot was probed for eEF2, Pgk1, Rps8 and Rpl10. Full-length western blots are presented in Supplementary Fig. S1. (d) Polysome profile analyses of the P_MET25-YEF3 strain grown in the presence and absence of 0.5 mM methionine. Before harvesting, the cells were either treated with 100 µg/ml cycloheximide for 10 minutes (+CHX) or left untreated (–CHX, ‘run-off conditions’). Whole cell extracts were resolved on sucrose gradients and the absorbance at 260 nm was measured during fractionation. The profiles are normalised to total area under the curve excluding non-ribosomal top fraction and are representatives of two biological replicates.