Abstract
A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input–output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.
Highlights
Numerous strategies have emerged for controlling levels of gene expression in yeast
In the 30-UTR, higher A/T content upstream of the polyadenylation site correlates with higher protein expression [7]
Z3EV is constitutively expressed from the ACT1 promoter, followed sequentially by URA3, the synthetic promoter to be tested and Green fluorescent protein (GFP) (Figure 1)
Summary
Numerous strategies have emerged for controlling levels of gene expression in yeast. These include introducing sequences that disfavor nucleosome localization near the transcription start site [1], varying both the affinity and number of binding sites for transcriptional activators in the promoter [2,3,4] and randomization of core promoter elements [5]. À3 of the gene’s initial ATG favor high protein expression, while out-of-frame AUGs in the 50-UTR near the start codon reduce protein levels [6]. While promoter strength and protein expression can be tuned in a variety of ways, it is desirable to selectively introduce or remove selected gene products so that the effects of gain or loss of function can be assayed [3,8,9,10]
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