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Fig. 1 | Genome Biology

Fig. 1

From: Distance-dependent inhibition of translation initiation by downstream out-of-frame AUGs is consistent with a Brownian ratchet process of ribosome scanning

Fig. 1

Testing PIC scanning models using thousands of dATG variants. A Predictions of the strictly unidirectional model and the Brownian ratchet scanning model on translational efficiency when an AUG is inserted downstream of the aAUG. B High-throughput construction of dATG variants with doped nucleotides and detection of GFP intensity en masse via FACS-seq. C Nomenclature of Solo and Duo variants. The aATGs, in-frame dATGs, and out-of-frame dATGs are shown in green, orange, and blue, respectively. Each dot in the sequences represents a nucleotide that cannot form an ATG triplet or an in-frame stop codon. D Boxplot shows GFP intensities (normalized by dTomato intensity, here and elsewhere in this study when presenting FACS-seq data) for Solo and Duo variants. P values were given by the Mann-Whitney U tests. E The average GFP intensities (dots) and the 95% confidence intervals (error bars) of Duo variants. The orange dashed line represents the average GFP intensity of all Duo variants with an in-frame dATG, and the blue curve represents the local regression line generated by R function “geom_smooth” (span = 1) for the Duo variants with an out-of-frame dATG. Duo variants with the dATG at positions from +4 to +6 (shown in gray) had fixed nucleotides at the −3 position of the dATG (due to the aATG), and therefore, were not used to fit the local regression line. F A small-scale experiment that introduced dATGs by synonymous mutations and strictly controlled the flanking sequences of the aATG and dATG. The GFP/dTomato fluorescence ratio estimated for each replicate is shown in a dot and the average value is shown in the red line. The GFP/dTomato ratios were normalized to the variant lacking additional dATG, which were 0.41, 0.64, 0.93, and 0.94 for the variants with dATGs inserted at the +8, +14, +20, and +26 positions, respectively. P values were given by t-tests

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