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Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.

Gnanasundram SV, Koš M - Mol. Biol. Cell (2014)

Bottom Line: Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems.A depletion time of <40 min was sufficient to achieve a robust phenotype.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.

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Northern blot analysis of HAS1 mRNA levels during depletion. Total RNA was isolated at indicated time points after addition of doxycycline. The blots were hybridized with probes complementary to HAS1 mRNA and SCR1 RNA (loading control).
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Figure 3: Northern blot analysis of HAS1 mRNA levels during depletion. Total RNA was isolated at indicated time points after addition of doxycycline. The blots were hybridized with probes complementary to HAS1 mRNA and SCR1 RNA (loading control).

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).


Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.

Gnanasundram SV, Koš M - Mol. Biol. Cell (2014)

Northern blot analysis of HAS1 mRNA levels during depletion. Total RNA was isolated at indicated time points after addition of doxycycline. The blots were hybridized with probes complementary to HAS1 mRNA and SCR1 RNA (loading control).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325845&req=5

Figure 3: Northern blot analysis of HAS1 mRNA levels during depletion. Total RNA was isolated at indicated time points after addition of doxycycline. The blots were hybridized with probes complementary to HAS1 mRNA and SCR1 RNA (loading control).
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).

Bottom Line: Various approaches using different repressible promoters, inducible degrons, or their combinations were developed.The target protein does not require a tag, and its elimination is several fold faster compared with standard promoter shutoff systems.A depletion time of <40 min was sufficient to achieve a robust phenotype.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.

Show MeSH