Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.
Bottom Line: A protein depletion by promoter shutoff or protein destabilization is an important tool in investigation of functions of essential genes.Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.A depletion time of <40 min was sufficient to achieve a robust phenotype.
Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.Show MeSH
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Mentions: The apparent longer than expected half-life could also be caused by an ongoing synthesis of the Has1p due to a delayed shutdown of its gene transcription and/or stability of its mRNA. However, the Northern blot analysis showed that, upon addition of doxycycline, the TetO7 promoter was rapidly repressed and the Has1p mRNA was undetectable after 20 min for all constructs (Figure 3). Therefore the contribution of the ongoing de novo synthesis of Has1p is limited. Nevertheless, we tried to improve the depletion efficiency by introducing a tetracycline-responsive (tc) aptamer into the mRNA upstream of the initiation AUG (Figure 1A). Such an aptamer has been described as blocking translation upon binding of tetracycline (Kötter et al., 2009). This should have allowed us not only to repress the transcription of the target gene but also to directly block its translation by addition of both doxycycline and tetracycline to the media. However, inclusion of the tc aptamer did not further improve the depletion rate, and neither the growth nor the rate of Has1p protein depletion was significantly affected (Figure 2C).
Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.