uORF3 is required for translational regulation of GCN4 expression. (A) The start codons of each individual uORF were mutated in GCN4-Luc and are indicated as crosses in the schematic. Fold-induction is indicated in brackets. (B) Pairs of uORFs were mutated as indicated. White-colored bars denote values obtained from nonstressed cells; black-colored bars, values obtained from cells treated with 15 mM 3AT for 3 h. (C) The 3AT sensitivity of a gcn4 mutant is complemented by reintroduction of wild-type GCN4 and a GCN4 construct containing uORF3 alone. The wild-type strain and gcn4 mutant are shown containing the empty vector. Strains were grown to stationary phase and diluted cultures (A600 = 1.0, 0.1, and 0.01) spotted onto SCD agar plates containing 1 mM 3AT.
