A simple LSM explains half of the variance of gene expression from natural yeast TLs. (A) Schematic describing a simple LSM. (Top) mRNA containing two AUG start codons. (Middle) The MCM predicts YFP expression using the Kozak strength for the main CDS start codon. (Bottom) The LSM predicts YFP expression using the Kozak strengths of all AUGs. This example shows the ribosome skipping the first AUG and initiating at the second ORF. The probability of initiating at YFP is given by the fraction of ribosomes that reach the CDS start codon (Pskip) times the Kozak strength at the YFP start codon (Pinit2). (B) The MCM explains 21.6% of the variance in YFP levels for TLs without any uAUGs (R² = 0.216; see Fig. S6 for uORF TLs). (C) Boxplots representing the distribution of measured YFP expression (y-axis) for native 5′ TLs binned by the number of uORFs (x-axis). Additional uAUGs further repress YFP expression. P-values: ***P < .001, **P < .01, *P < .05. uAUGs: n = 0:9058, 1:1448, 2:309, 3:126, 4:50, 5:22, 6:10, 7:4 (D) Linear regression model of measured YFP (y-axis) versus the LSM predicted Kozak strength (x-axis) for each 5′ TL.