Fast protein-depletion system utilizing tetracycline repressible promoter and N-end rule in yeast.
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.
Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.Show MeSH
Mentions: The results presented so far show that growth in the strains carrying the TetO7-Ubi-Leu cassette slows several hours before growth in the strains carrying the standard TetO7 promoter. While convenient, monitoring of cell growth allows only a gross measure of the defects resulting from the reduced levels of the protein studied. The change in the growth rate cannot be used to reliably time the first appearance of a phenotype resulting from the target protein depletion. Therefore we took advantage of the easily detectable RNA-processing phenotypes of Has1p and Prp19p and determined the time of appearance of first RNA-processing defects in the depleted strains. The Has1p protein is essential for rRNA processing, in particular for production of the 20S pre-rRNA intermediate (Emery et al., 2004). Therefore we used Northern blotting to determine the shortest depletion time required to abolish 20S pre-rRNA synthesis. Surprisingly, only 40 min after the start of depletion, the 20S pre-rRNA levels were clearly reduced and synthesis of 20S was barely detectable after 60 min in the TetO7-Ubi-Leu-Has1 strain (Figure 5A). The inclusion of the tc aptamer had only a mild positive effect on the depletion (Figure 5A, bottom panel). In comparison, in the standard TetO7-Has1 strain, the reduction in the 20S pre-rRNA levels was noticeable after only 240 min, with the 20S pre-rRNA signal comparable with the 40-min time point in the TetO7-Ubi-Leu-Has1 strain. Therefore the depletion time required to achieve a robust measurable phenotype was reduced sixfold in the TetO7-Ubi-Leu-Has1 strain. The Prp19p is an essential splicing factor. Northern blotting was used to detect unspliced, intron-containing U3 small nucleolar RNA (snoRNA) at different time points during depletion (Figure 5B). It is important to note that the unspliced U3 snoRNA (similar to unspliced pre-mRNAs) can also be detected in normally growing wild-type cells, explaining the weak signal present in all the analyzed time points. Increased levels of the unspliced U3 were detected after 1–2 h in the TetO7-Ubi-Leu-Prp19 strain compared with 8 h in TetO7-Prp19 strain. Thus, also in the case of Prp19p, the appearance of the first processing defects resulting from the protein depletion is largely accelerated.
Affiliation: Biochemistry Center, University of Heidelberg, 69120 Heidelberg, Germany.