Fig. 3.

eIF1 overexpression inhibits initiation on poorly recognized codons. The results are from dual luciferase measurements. Renilla luciferase is initiated by an AUG in a consensus (good) context and is used to normalize firefly luciferase values. The firefly reporter is initiated by the codon, and the context, indicated on the left. (A and B) Firefly luciferase measurements from cells cotransfected with (i) empty vector (control)—gray-blue bars; (ii) “10×” vector expressing eIF1 in its native initiation context—maroon bars; or (iii) “10×” vector expressing eIF1 in the near consensus (good*) context—yellow bars. Right-facing bars show relative expression of firefly and Renilla luciferase. Left-facing bars show the ratio of normalized firefly luciferase expression (fold repression) calculated by comparing the values of (i) cells transfected with empty vector (control) and cells transfected with vector containing eIF1 initiated with the wild-type context (green bars); or (ii) cells transfected with empty vector and cells transfected with vector containing eIF1 initiated with the good* context (orange bars). (A) Results from experiments varying the context at positions −3 and +4. Varied nucleotides are shown in green or red, depending on whether they are predicted to provide a good or a poor context for initiation, respectively. (B) Results from experiments changing the start codon to one of the nine possible near cognate non-AUG codons. The fold repression marked by “◆” is probably an underestimate because the firefly activity is close to background level even under control conditions. The results in A and B represent independent experiments accounting for the slight difference in the values from firefly luciferases starting with “best” and “eIF1” contexts.