116 6 Constitutive and Regulated Promoters in Yeast
The cytochrome c isoform 1 (CYC1) promoter has been extensively charac-
terized [1, 16]. This gene is involved in cell respiration and its transcription is
triggered by heme [16, 30]. When this metabolite is present in the cells, it
binds the transcription activator Hap1, which can then recognize its cognate
TFBS [30, 85]. Today, a truncated version of this promoter is used to drive
mild and relatively constant expression in fermentative growth conditions.
For this reason the CYC1 promoter is usually described as a constitutive
promoter [9].
Besides genes involved in energy metabolism, those involved in other basic
tasks, like cell shape maintenance and translation, also have constitutive promot-
ers. For example, the promoter of the gene encoding β‐actin (ACT1) displays a
combination of regulatory elements ensuring a constant transcription in both
fermentative and non‐fermentative growth conditions [86]. Similarly, the
translation elongation factor EF‐1 α (TEF1) has a promoter ensuring approxi-
mately stable expression during all growth phases and in media containing differ-
ent carbon sources [76, 80, 87].6.4 Synthetic Yeast Promoters
A synthetic promoter carries nonnative sequences. We describe two main groups
of synthetic promoters. One includes modified versions of natural promoters,
and the other contains hybrid promoters. As illustrated below, the main differ-
ence between promoters belonging to each class is the strategy used to construct
them.6.4.1 Modified Natural Promoters
The systematic modification of a promoter leads to a library spanning a wide
range of transcription initiation frequencies. Within this library, each member
drives transcription initiation with a specific strength. Since the members of the
library are derived from a single promoter, they share similar regulatory features;
that is, they respond to the same stimulus [88–90]. There are two main methods
for obtaining promoter libraries: either by introducing point mutations or by
substituting short sequences with randomized oligonucleotides (reviewed in
[91]) (Figure 6.1).
Mutations in essential regulatory sequences are likely to cause a substantial
change in activity, because they can alter the binding affinity of the cognate
proteins. A library of TEF1 promoter variants was obtained by error‐prone
PCR. With this approach the point mutations spanned along the complete
sequence of the promoter [92]. The library covered a range of activities from
8% to 120% relative to the native TEF1 promoter [93]. A variation of this strat-
egy consists in limiting the point mutations to specific regions of the promoter,
for example, to the TATA element. These modifications alter the efficiency of
the PIC assembly [94]; therefore they affect the overall performance of the
promoter [23].