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. 2004 Jul 26;101(31):11269–11274. doi: 10.1073/pnas.0400541101

Fig. 4.

Fig. 4.

uORF1 functions as a positive regulator, and uORF2 is inhibitory in a scanning mechanism that regulates the translation of ATF4 mRNA. Schematic representation of the WT and different mutant versions of the ATF4-leader sequences fused to luciferase are shown to the left of each luciferase measurement. (A) A box represents the WT version of uORF 1 and uORF2, and an X indicates a nonfunctional uORF due to a mutation in the initiation codon. S/S and A/A MEF cells were cotransfected with the indicated ATF4-Luc plasmid and a control Renilla luciferase plasmid. The transfected cells were treated with Tg for 6 h (gray and black bars) or no ER stress agent (white and stippled bars). Relative light units (RLU) is a ratio of firefly luciferse units normalized for Renilla luciferase units, and each value was derived from three independent transfections. For clarity the histogram is represented in two different scales. (B) Three stem–loop structures with the indicated ΔG values in kcal/mol were inserted between uORF1 and uORF2 in the WT ATF4-Luc construct. Alternatively, the stem–loop structures were inserted in ATF4-leader regions containing an uORF1 mutation (C) or an uORF2 mutation (D). (E) A 120-bp sequence was inserted in the ATF4-leader region between uORF1 and uORF2. The transfected S/S MEF cells were treated with Tg for 6 h (gray bar) or no ER stress agent (white bar), and the RLU was measured as described for A.