Figure S2.

Experimental Validation of Roles of Initiation and Elongation Rates on Protein Production in Yeast, Related to Figures 2 and 4

(A and B) The relationship between protein production, codon bias, and 5′ mRNA folding for 141 synthetic GFP genes expressed in Saccharomyces cerevisiae. We constructed 141 GFP genes, all encoding the same protein sequence but varying randomly at synonymous sites, as previously described (Kudla et al., 2009). BY4741 yeast was transformed with a Gal-induced 2-micron plasmid, then grown for 24h in 2% Raf +2% Gal. Fluorescence was then measures by FACS. Measurements typically represent three biological replicates, on the same day. Panel (A) shows the relationship between fluorescence and each GFP gene’s yeast Codon Adaptation Index; Panel (B) shows the relationship between fluorescence and each GFP gene’s predicted 5′ mRNA folding. 5′ mRNA folding was estimated using mfold, as previously described (Kudla et al., 2009). There is no significant correlation between expression level and codon bias, suggesting that elongation rates do not modulate protein production in these constructs. By contrast, there is a significant correlation between expression level and mRNA folding near the translation initiation site, indicating that initiation rates strongly influence protein production in these constructs.