114 6 Constitutive and Regulated Promoters in Yeast
the two open reading frames revealed the presence of a galactose‐dependent
UAS [54, 55]. The UAS contains two shared TFBSs bound by the transcriptional
activator Gal4. These TFBSs are sufficient to confer galactose‐dependent regula-
tion to a promoter [31]. The first yeast synthetic hybrid promoter, discussed
above, was assembled by placing the GAL1–10 UAS upstream of the core pro-
moter region of another gene. The construct exhibited the typical GAL1–10
galactose‐dependent regulation [3].
The promoter of the acid phosphatase (PHO5) contains two homologous
TFBSs bound by the transcription activators Pho4 and Pho2 when inorganic
phosphate is depleted in the culture medium. The presence of inorganic phos-
phate in the medium leads to Pho4 sequestration in a protein complex that
does not allow its binding to the PHO5 promoter [56, 57]. Although this
promoter displays some basal activity in the repressed state [56], it has been
successfully used to express heterologous genes, like the hepatitis B surface
antigen [58].
The promoters of genes involved in copper metabolism are frequently used for
driving transcription of heterologous genes [59, 60]. The CUP1 promoter is stim-
ulated by copper [61]. Its activation depends on the transcription activator Ace1,
whose ability to bind its TFBSs is controlled by its interaction with copper ions
[62]. Although this is a popular promoter, its use is limited to strains carrying the
wild‐type CUP1 locus. With this genetic background it is possible to avoid toxic
effects related to excess copper, since the CUP1 gene encodes a metallothionein
acting as a copper chelator. However, copper is essential in biological processes
like respiration; therefore it is usually present in traces in culture media. This
small amount of copper causes a substantial basal expression of genes under the
control of the CUP1 promoter. The metallothionein encoded by the wild‐type
CUP1 locus contributes to lowering the amount of available copper. As a conse-
quence the basal activity of the heterologous construct is lowered [63]. An excess
of copper prevents the transcription of genes encoding copper transporters, like
CTR1 and CTR3 [64]. The promoters of these genes contain specific TFBSs
bound by the transcription activator Mac1. When Mac1 interacts with copper
ions, its DNA‐binding and activation activities are inhibited [65]. A collection of
expression vectors containing CUP1, CTR1, and CTR3 promoters is available for
coordinated induction and inhibition experiments [66].
Regulation of the promoters described so far depends on a single TFBS.
However, some promoters display a combination of TFBSs bound by different
transcription factors. This results in more sophisticated regulation. Several
examples are described below.
The promoter of DAN1, a mannoprotein, contains a set of TFBSs bound by
both activators and repressors. The combination of the activity of these tran-
scription factors results in complete repression in the presence of oxygen and full
activation when this gas is absent from the culture medium [67]. For induction,
this promoter requires stringent anaerobiosis, which can be realized by bubbling
nitrogen in the cultures. However, this experimental setup is not convenient for
large scale overexpression experiments. As such, random mutagenesis of the
DAN1 promoter yielded variants less sensitive to oxygen that can be induced in
microaerobiosis [68].