Promoters are fundamental components of synthetic gene circuits. They are DNA segments where transcription initiation takes place. New constitutive and regulated promoters are constantly engineered in order to meet the requirements for protein and RNA expression into different genetic networks. In this work, we constructed and optimized new synthetic constitutive promoters for the yeast Saccharomyces cerevisiae. We started from foreign (e.g., viral) core promoters as templates. They are, usually, unfunctional in yeast but can be activated by extending them with a short sequence, from the CYC1 promoter, containing various transcription start sites (TSSs). Transcription was modulated by mutating the TATA box composition and varying its distance from the TSS. We found that gene expression is maximized when the TATA box has the form TATAAAA or TATATAA and lies between 30 and 70 nucleotides upstream of the TSS. Core promoters were turned into stronger promoters via the addition of a short UAS. In particular, the 40 nt bipartite UAS from the GPD promoter can enhance protein synthesis considerably when placed 150 nt upstream of the TATA box. Overall, we extended the pool of S. cerevisiae promoters with 59 new samples, the strongest overcoming the native TEF2 promoter.

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Journal Article

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Feng X, Marchisio MA

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