Novel S. cerevisiae Hybrid Synthetic Promoters Based on Foreign Core Promoter Sequences

Abstract

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.

Logic Circuits Based on 2A Peptide Sequences in the Yeast Saccharomyces cerevisiae

“…In the other TU, yEGFP expression was controlled by a synthetic promoter. In order to design this new synthetic promoter, we considered that (1) the maximal core promoter activity is achieved when the TATA box and the TSS are separated by, roughly, 30 up to 70 nucleotides; (2) operator position influences both promoter strength and repression efficiency; and (3) greater repression by TetR was observed when its operators were placed close to the TATA box rather than the TSS . Thus, we realized the synthetic promoter “pCYC1min_tetOp­(−57,19 nt)_1 × TATA(−71)” by placing the only TATA box along positions −71 and −65 upstream of the TSS (+1 position) and adding a tetOp (19-nt long) between position −57 and −39, i.e., only 8 nt downstream of the TATA box (Figure A).…”

Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol

“…Thus, we modified the receptor part of biosensors 1 by employing two weaker promoters to express the chimeric activator (still carrying VP64). We chose the synthetic promoter DEG1t_pCYC1noTATA (19.75% as strong as pGPD 25 ) and the viral CMV promoter (pCMV, 4.74% as pGPD 26 —see Table S1 ). With respect to pGPD, the expression of the synthetic activator decreased drastically (see Fig.…”

Synthetic polycistronic sequences in eukaryotes

“…In contrast, many synthetic promoters of different, well-characterized strength, were developed over the last four decades, at least. They permit to tune, rather precisely, the amounts of proteins in a circuit through various expedients such as: assembling multiple UASs (upstream activating sequences) with different core sequences [ 124 ]; nucleosome removal [ 125 ]; insertion of intron sequences along the 5′UTR [ 126 ]; mutations in the TATA box sequence; and varying the distance between the same TATA box and either the TSS (transcription start site) or the UASs [ 127 ]. Moreover, longer polycistronic sequences might be obtained in the future by combining 2 A peptides with bidirectional synthetic promoters [ 116 , 117 ].…”