120 6 Constitutive and Regulated Promoters in Yeast
domain of Gal4 are separated and fused to the chromoprotein phytochrome
PhyB and its interactor Pif3, respectively. Red light converts the PhyB fusion
into its active form, which interacts with the Pif3 fusion, activating transcription.
Far‐red light converts the PhyB fusion into its inactive form, which cannot bind
Pif3, disabling transcription initiation [126].
The activity of heterologous transcription factors can be precisely controlled
by fusing additional domains that, for example, trigger nuclear localization upon
a specific stimulus. The human estrogen receptor, when fused to a transcription
activator, confers a hormone‐dependent regulation. This chimera triggers tran-
scription initiation only when β‐estradiol is added to the culture medium [102].
Binding of the hormone to the estrogen receptor causes the nuclear localization
of the transcription activator, which, in the absence of inducer, is diffusing all
over the cell [127]. An activator containing the Gal4 DNA‐binding domain and
the estrogen receptor binds GAL promoters, but its activity does not depend on
the carbon source [127, 128]. Estrogen‐regulated activators based on heterolo-
gous DNA‐binding domains such as LexA or synthetic zinc fingers result in
orthogonal systems that specifically regulate the expression of the target pro-
moters [100, 102]. The LexA‐based activator induces the expression of the target
gene in different growth conditions. Its overall activity can be finely tuned with
the concentration of β‐estradiol in the culture medium, the number of LexA
TFBSs in the target promoter, and the choice of the activation domain [102].
An essential aspect of regulated synthetic promoters is the tightness of their
regulation. A promoter is tightly regulated when it does not have any basal activ-
ity in the absence of the stimulus. The basal activity of some promoters depends
on the residual activity of the transcription activator in the absence of the stimu-
lus [114]. Alternatively, the basal expression can be the consequence of ectopic
transcriptional events starting upstream of the promoter itself [20]. In this case,
the insulation of the synthetic transcription unit is necessary. This can be
obtained by placing a transcriptional terminator in front of the synthetic
promoter [129].
Regulation of gene expression can also be achieved by repression of transcrip-
tion initiation. A protein binding the DNA between the upstream element and
the core promoter or within the core promoter prevents the establishment of the
interactions needed for the effective recruitment of the PIC, causing transcrip-
tion repression by steric hindrance [33, 36, 89, 107, 130]. The DNA‐binding pro-
tein tetR was used to systematically study this kind of repression. A collection of
GAL1 promoter variants containing different number of tetR TFBSs placed
between the TATA element and the TSS was tested. It was observed that increas-
ing the number of such TFBSs reduced the basal expression of the system.
Moreover, repression was stronger when the TFBSs were placed in close proxim-
ity to the TATA element [103, 105]. At intermediate levels of induction, the
expression levels of the genes targeted by tetR showed a broad cell‐to‐cell varia-
bility. Reduction of such a cell‐to‐cell variability was obtained by placing tetR
expression under its own control, implementing a negative feedback loop [131].
Te tR expression under negative feedback control also resulted in a “linearized”
dose–response curve, allowing for larger concentration ranges of tetracycline
and therefore better titratability. This negative feedback‐based concept has also