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. 2017 Aug 21;11:25.
doi: 10.1186/s13036-017-0068-1. eCollection 2017.

Nucleotides Upstream of the Kozak Sequence Strongly Influence Gene Expression in the Yeast S. cerevisiae

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Free PMC article

Nucleotides Upstream of the Kozak Sequence Strongly Influence Gene Expression in the Yeast S. cerevisiae

Jing Li et al. J Biol Eng. .
Free PMC article

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: 5′-UTR; Kozak sequence; S. cerevisiae; Synthetic biology.

Figures

Fig. 1
Fig. 1
Effect of point mutations in the extended Kozak sequence on fluorescence expression. Fluorescence levels are plotted relative to k 1 (a) and k 0 (b). Control corresponds to a yeast strain without the yEGFP gene. The nucleotide that replaced an adenine in k 1 and the position at which the mutation took place are given below the name of each synthetic leader sequence. Asterisks, p-value <0.05 vs. k 1 (a) or k 0 (b)
Fig. 2
Fig. 2
Multiple point mutations to guanine. The ratio between the fluorescence level of the synthetic 5-UTRs from k 26 to k 38 and that of k 1 are reported. The number of adenines or guanines in the upstream region is given below the leader sequence name (from k 27 to k 38). The subscripts −1, −2, and −3 indicate that an adenine is present in the extended Kozak sequence only at the corresponding position. Subscript i represents intermixed (see main text). Asterisks, p-value <0.05 vs. k 1
Fig. 3
Fig. 3
Effect of point mutations in the upstream region on fluorescence relative to k 1. The nucleotide that replaced an adenine in k 1 and the position at which the mutation took place are given below the name of each synthetic leader sequence. Asterisks, p-value <0.05 vs. k 1
Fig. 4
Fig. 4
mRNA secondary structures. a A giant hairpin is present in the mRNA secondary structure corresponding to the MFE of both k 0 and k 1. The hairpin loop contains the −15…−1 region. The portion of the 5-UTR in our analysis is free from any pairing interactions in its wild-type configuration (k 0) and in that theoretically optimized for high protein expression (k 1). The loop of the giant hairpin is reduced in k 4 owing to the base-pairing interaction between the guanine at position −1 and the cytosine at position −31. In every mRNA structure presented, a green arrow indicates position +1, and a red arrow indicates position −15. b The disruption of the giant hairpin induces a decrease in the MFE of the mRNA secondary structure. k 26 and k 31 are associated with the lowest MFEs computed in our analysis. The two sequences contain multiple guanines in the extended Kozak sequence involved in pairing interactions with the CDS. A similar pattern is also present in k 30. Here, however, a second mini-loop around the START codon provokes an increase in MFE. The MFE of k 26 is substantially lower than those of k 30 and k 31 because of the presence of another stem due to pairing interactions between the upstream region and the CYC1 terminator. Nevertheless, the fluorescence levels of k 30 and k 31 are only approximately 1.2-fold higher than that of k 26
Fig. 5
Fig. 5
Low MFE values are associated with reduced fluorescence expression. Red bars, difference between MFEs of the corresponding 5-UTR and k 1 (ΔMFE). Blue bars, 10-fold magnified ratio between the fluorescence level of the indicated 5-UTR and that of k 1. Apart from k 1, sequences are sorted by increasing ΔMFE. All sequences except k 4 contain multiple point mutations with respect to k 1. Asterisks above blue bars, p-value <0.05 vs. k 1
Fig. 6
Fig. 6
mRNA secondary structures. a k 27 differs from k 29 only by a guanine instead of an adenine at position −1. However, their mRNA secondary structures are dissimilar. In k 27, the extended Kozak sequence is involved in base-pairing interactions with the CYC1 terminator, whereas in k 29 the extended Kozak sequence is locked into a stem with the CDS. The MFE associated with k 27 is lower than that of k 29, but there is no difference between the fluorescence levels of the two sequences (p-value =0.20). b Multiple guanines in the upstream region give rise to mRNA structures characterized by base-pairing interactions between the 5-UTR and the CYC1 terminator. k 28 and k 34 have six guanines in a stem with the CYC1 terminator, whereas k 35 has only 5 guanines in an analogous structure. This causes an increase in MFE and consequently a higher fluorescence

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References

    1. Endy D. Foundations for engineering biology. Nature. 2005;438(7067):449–53. doi: 10.1038/nature04342. - DOI - PubMed
    1. Carothers JM, Goler JA, Juminaga D, Keasling JD. Model-driven engineering of RNA devices to quantitatively program gene expression. Science. 2011;334(6063):1716–9. doi: 10.1126/science.1212209. - DOI - PubMed
    1. Kozak M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo. Nature. 1984;308(5956):241–6. doi: 10.1038/308241a0. - DOI - PubMed
    1. Tuller T, Ruppin E, Kupiec M. Properties of untranslated regions of the S. cerevisiae genome. BMC Genomics. 2009;10(1):391. doi: 10.1186/1471-2164-10-391. - DOI - PMC - PubMed
    1. Lubliner S, Keren L, Segal E. Sequence features of yeast and human core promoters that are predictive of maximal promoter activity. Nucleic Acids Res. 2013;41(11):5569–81. doi: 10.1093/nar/gkt256. - DOI - PMC - PubMed

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