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.

Keywords: Saccharomyces cerevisiae; TATA box; UAS; gene expression; promoters; synthetic biology.

Figure 1

Promoters in S. cerevisiae. (A) The structure of promoters in the baker’s yeast is characterized by four main elements: The upstream activating sequence (UAS), the TATA box (where RNA polymerase II—RNA pol II—binds), the transcription start site (TSS), and the 5′UTR (untranslated region). The core promoter sequence consists of the TATA box(es), the TSS(s), and the 5′UTR. (B) The pCYC1noTATA begins at position −16 with respect to the TSS at position +1 in the CYC1 yeast promoter. It contains six TSSs over 43 of the 71 nucleotides that constitute the CYC1 promoter 5′UTR.

Figure 2

Fluorescence expression from foreign promoters and yeast synthetic core promoters. (A) Full or reduced foreign promoter sequences appear either unfunctional (pSV40full, pSV40r) or extremely weak if compared to the strong yeast constitutive GPD promoter. Fluorescence intensity, on the y-axis, is represented in a logarithmic scale and expressed in arbitrary units (AU). (B) Synthetic core promoters constructed by extending foreign promoters with pCYC1noTATA show a dramatic enhancement in fluorescence expression. (C) Comparison of the mean fluorescence expressed by our eight new synthetic core promoters. Each mean fluorescence value comes from three independent experiments.

Figure 3

Constructing yeast synthetic core promoters via mutations on the TATA box. (A) Twenty-six core promoters have been realized via mutation(s) on the TATA box of pSV40core*(TATAAAA-90). They were distributed into eight groups depending on their strength. Groups were determined by both the two-sided Welch t–test and one-way ANOVA. (B) Effects of T → A mutation at different TATA box positions. Each mean fluorescence value comes from three independent experiments.

Figure 4

Effects on fluorescence expression due to the distance between the TATA box and the TSS. Experiments were carried out (in three different days) on variants of pSV40*(TATATAT). The symbol ° on top of different columns indicates that there is no significant statistical difference among the fluorescence levels of the corresponding promoters (p-value = 0.3704, one-way ANOVA).

Figure 5

Enhancement in the fluorescence level of pSV40*(TATATAT-49) due to the presence of an UAS. (A) Most of the UASs considered in this work had a similar effect when placed 150 nt upstream of pSV40*(TATATAT-49). The symbol ° on top of five columns indicates no significant statistical difference (p-value = 0.1005, one-way ANOVA) among the corresponding fluorescence levels. (B) Variation in fluorescence expression due to different spacers between UAS_GPD(40nt) and pSV40*(TATATAT-49). The configuration with a 150 nt-long spacer was the most effective though 30 nt were enough to have a statistically significant increase in the fluorescence level of pSV40*(TATATAT-49). Each mean fluorescence value is the result of three independent experiments; * corresponds to a p-value < 0.05; ** to a p-value < 0.01 (two-sided Welch’s t-test).

Figure 6

Variation in the fluorescence level of foreign promoters upon addition of an UAS. (A) A 150 nt spacer between UAS_GPD(40nt) and MLPcore* drastically improved fluorescence expression, whereas a 30 nt spacer had no significant effects (NS: p-value = 0.1961, two-sided Welch’s t-test). (B) The weak pTKcore*, modified with the strong TATA box—TATAAAA, underwent dramatic enhancement in fluorescence expression by placing UAS_GPD(40nt) both 30 and 150 nt upstream of the TATA box. Each mean fluorescence value comes from three independent experiments, NS: No significant statistical difference.