Figure 4.

The eIF4G1-1 mutant imparts a modest Gcn^– phenotype and specifically increases the efficiency of REI past the uORF1 translation in vivo. (A) Schematic showing the GCN4-LacZ construct with all four upstream uORFs. (B) The eIF4G1-1 and -459 mutants impart the Gcn^– phenotype. Along with pMP121 (containing the wt GCN4 leader), the GCN4-lacZ construct from panel A was introduced individually into the strains carrying various eIF4G-encoding alleles described in Figure 3. The resulting transformants were pre-cultured in minimal media overnight, diluted to OD₆₀₀ ∼0.3, grown for 2 h., and the same aliquots of cultures were either treated with 10 mM 3-AT (to induce the GCN4-lacZ expression) or without 3-AT for 6 h. The β-galactosidase activities were measured in the WCEs and expressed in units of nmol of o-nitrophenyl-b-d-galactopyranoside hydrolyzed per min per mg of protein. The mean values and standard deviations obtained from at least three independent measurements with three independent transformants, and activity in the mutant constructs relative to wt (in %), as well as the induced vs. induced fold-increases are given where applicable. Statistical significance of the differences between wt and mutant eIF4G1 alleles was assessed using Anova: Single Factor test with Post Hoc testing (*P< 0.05). (C) Schematics showing various modified GCN4-LacZ constructs described in the main text. (D) The eIF4G1-1 mutant specifically increases the efficiency of REI past the uORF1 translation in vivo. The GCN4-lacZ constructs from panel C were introduced individually into the strains carrying various eIF4G-encoding alleles described in Figure 3. The resulting transformants were pre-cultured in minimal media overnight, diluted to OD₆₀₀ ∼0.35, grown for an additional 6 hrs. and the β-galactosidase activities were measure and analyzed as described in panel B (*P< 0.05).