Nucleotides upstream of the Kozak sequence strongly influence gene expression in the yeast S. cerevisiae

In the yeast Saccharomyces cerevisiae, as in every eukaryotic organism, the mRNA 5'-untranslated region (UTR) is important for translation initiation. However, the patterns and mechanisms that determine the efficiency with which ribozomes bind mRNA, the elongation of ribosomes through the 5'-UTR, and the formation of a stable translation initiation complex are not clear. Genes that are highly expressed in S. cerevisiae seem to prefer a 5'-UTR rich in adenine and poor in guanine, particularly in the Kozak sequence, which occupies roughly the first six nucleotides upstream of the START codon.We measured the fluorescence produced by 58 synthetic versions of the S. cerevisiae minimal CYC1 promoter (pCYC1min), each containing a different 5'-UTR. First, we replaced with adenine the last 15 nucleotides of the original pCYC1min 5'-UTR-a theoretically optimal configuration for high gene expression. Next, we carried out single and multiple point mutations on it. Protein synthesis was highly affected by both single and multiple point mutations upstream of the Kozak sequence. RNAfold simulations revealed that significant changes in the mRNA secondary structures occur by mutating more than three adenines into guanines between positions -15 and -9. Furthermore, the effect of point mutations turned out to be strongly context-dependent, indicating that adenines placed just upstream of the START codon do not per se guarantee an increase in gene expression, as previously suggested.New synthetic eukaryotic promoters, which differ for their translation initiation rate, can be built by acting on the nucleotides upstream of the Kozak sequence. Translation efficiency could, potentially, be influenced by another portion of the 5'-UTR further upstream of the START codon. A deeper understanding of the role of the 5'-UTR in gene expression would improve criteria for choosing and using promoters inside yeast synthetic gene circuits.