Fig. 4

The distance-dependent inhibitory effect of out-of-frame uAUGs on translation initiation at the aAUG. A Predictions of the Brownian ratchet scanning model on translational initiation at the aAUG, when an out-of-frame uAUG is introduced at various positions. B Solo and Duo variants in the uATG library. The aATGs, in-frame uATGs, and out-of-frame uATGs are shown in green, orange, and purple, respectively. Each dot in the sequences represents a nucleotide that cannot form an ATG triplet or an in-frame stop codon for the uATG (if exists). C The average GFP intensities (dots) and the 95% confidence intervals (error bars) of Duo variants with the uATG placed at positions ranging from −30 to −3 in the hoΔ (left panel) and the upf1Δ (right panel) backgrounds. The orange dashed line represents the average GFP intensity of all Duo variants with an in-frame uATG, and the purple curve represents the local regression line (span = 1) for the Duo variants with an out-of-frame uATG. Duo variants with the uATG at positions from −5 to −3 had fixed nucleotides at the −3 position of the aATG (due to the uATG) and Duo variants with the uATG at positions from −30 to −28 had fixed nucleotides at the −3 position of the uATG (due to the upstream flanking sequence). These dots are shown in gray and were not used to fit the local regression line. D A small-scale experiment that strictly controlled the flanking sequence of uATG and aATG. The GFP/dTomato fluorescence ratios were normalized to the variant lacking additional uATG. P values were given by t-tests