Experimental design and biological discovery. (A) Experimental design of a liquid-based growth assay of 489,348 5′ UTR variants. Random 50 nt were introduced directly upstream of the HIS3 coding sequence, replacing the 56 nt of the 5′ UTR of the CYC1 promoter. These constructs were introduced into a low copy number plasmid, transformed into yeast without a native copy of HIS3, and competed in media lacking histidine. The enrichment of each UTR after growth was measured by using massively parallel sequencing before and after selection. (B) 5′ UTR enrichment scores per nucleotide were averaged at each position. (C) The Kozak sequences (−5 to −1 position) leading to the highest His3 protein expression compared to the most abundant yeast Kozak sequence (AAAAA). (D) The enrichment of 5′ UTRs based on the predicted minimum free energy of the −50 to +70 sequences. (E) The enrichment of 5′ UTRs based on the presence of an upstream AUG (uAUG) and a stop codon within the UTR. Upstream open reading frames (uORFs) are characterized by an in-frame uAUG followed by a termination codon before the primary ORF start codon, or an out-of-frame uAUG followed by a stop codon before or after the primary ORF start codon.
