Fast-acting and nearly gratuitous induction of gene expression and protein depletion inSaccharomyces cerevisiae
Open Access
- 15 November 2011
- journal article
- Published by American Society for Cell Biology (ASCB) in Molecular Biology of the Cell
- Vol. 22 (22), 4447-4459
- https://doi.org/10.1091/mbc.e11-05-0466
Abstract
We describe the development and characterization of a system that allows the rapid and specific induction of individual genes in the yeast Saccharomyces cerevisiae without changes in nutrients or temperature. The system is based on the chimeric transcriptional activator Gal4dbd.ER.VP16 (GEV). Upon addition of the hormone β-estradiol, cytoplasmic GEV localizes to the nucleus and binds to promoters containing Gal4p consensus binding sequences to activate transcription. With galactokinase Gal1p and transcriptional activator Gal4p absent, the system is fast-acting, resulting in readily detectable transcription within 5 min after addition of the inducer. β-Estradiol is nearly a gratuitous inducer, as indicated by genome-wide profiling that shows unintended induction (by GEV) of only a few dozen genes. Response to inducer is graded: intermediate concentrations of inducer result in production of intermediate levels of product protein in all cells. We present data illustrating several applications of this system, including a modification of the regulated degron method, which allows rapid and specific degradation of a specific protein upon addition of β-estradiol. These gene induction and protein degradation systems provide important tools for studying the dynamics and functional relationships of genes and their respective regulatory networks.Keywords
This publication has 42 references indexed in Scilit:
- Mitochondrial Dysfunction Leads to Nuclear Genome Instability via an Iron-Sulfur Cluster DefectCell, 2009
- Efficient protein depletion by genetically controlled deprotection of a dormant N‐degronMolecular Systems Biology, 2009
- Single-RNA counting reveals alternative modes of gene expression in yeastNature Structural & Molecular Biology, 2008
- Frequency-modulated nuclear localization bursts coordinate gene regulationNature, 2008
- Membrane Localization of Scaffold Proteins Promotes Graded Signaling in the Yeast MAP Kinase CascadeCurrent Biology, 2008
- Structural Basis for Dimerization in DNA Recognition by Gal4Structure, 2008
- Genetic reconstruction of a functional transcriptional regulatory networkNature Genetics, 2007
- Identifying transcription factor functions and targets by phenotypic activationProceedings of the National Academy of Sciences of the United States of America, 2006
- GAL4-VP16 is an unusually potent transcriptional activatorNature, 1988
- Negative effect of the transcriptional activator GAL4Nature, 1988