Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity
Open accessAbstract
Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P tet promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2 S -M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range, as previously shown for the widely applied tTA.
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Random mutagenesis of tTA followed by a functional screen for the reverse, dox-inducible phenotype in S. cerevisiae resulted in rtTA-S2, with 4 amino acid substitutions in the TetR domain; two mutations (E19G and A56P) reversed the phenotype, while D148E and H179R enhanced activity.
“Random mutagenesis of tTA followed by a functional screen for the reverse, dox-inducible phenotype in the yeast S. cerevisiae resulted in a novel rtTA variant, rtTA-S2, with 4 amino acid substitutions in the TetR domain [15] (Fig. 2). Two of the selected mutations (E19G in the DBD and A56P in the helix connecting the DBD with the core) were shown to reverse the phenotype, whereas the other”
From Tet-On Systems For Doxycycline-inducible Gene Expression (2016) · cites this paper as [15]
A second round of mutagenesis and screening in S. cerevisiae yielded an additional DBD mutation (S12G) that increased the activity of rtTA-S2, in particular at low dox concentrations.
“A second round of mutagenesis and screening in S. cerevisiae yielded an additional DBD mutation (S12G) that increased the activity of rtTA-S2, in particular at low dox concentrations.”
From Tet-On Systems For Doxycycline-inducible Gene Expression (2016) · cites this paper as [15]
The rtTA2S-S2 and rtTA2S-M2 variants demonstrated increased stability and reduced background activity in the absence of dox when compared with the original rtTA.
“These studies resulted in the rtTA2S-S2 and rtTA2S-M2 variants that demonstrated increased stability and reduced background activity in the absence of dox when compared with the original rtTA.”
From Tet-On Systems For Doxycycline-inducible Gene Expression (2016) · cites this paper as [15]
The rtTA2S-M2 variant demonstrated the highest sensitivity toward dox and functioned at a 10-fold lower dox concentration than the original rtTA and rtTA-S2.
“The rtTA2S-M2 variant (a.k.a. Tet-On Advanced, Clontech) demonstrated the highest sensitivity toward dox and functioned at a 10-fold lower dox concentration than the original rtTA and rtTA-S2.”
From Tet-On Systems For Doxycycline-inducible Gene Expression (2016) · cites this paper as [15]
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- Authors
- Urlinger, S., Baron, U., Thellmann, M., Hasan, M. T., Bujard, H., & Hillen, W.
- Affiliations
- Heidelberg University (DE)
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2000-06-20
- Volume
- 97
- Issue
- 14
- Pages
- 7963-7968
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC16653
- OpenAlex
- W2150712887
- Cited by
- 928
- References
- 2
- Citations by year
- 2026: 12 · 2025: 18 · 2024: 11 · 2023: 24 · 2022: 33 · 2021: 19 · 2020: 25 · 2019: 19 · 2018: 29 · 2017: 26 · 2016: 29 · 2015: 33 · 2014: 32 · 2013: 40 · 2012: 46
- FWCI
- 22.82
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Max Planck Institute for Plasma Physics (submittedVersion)
- Max Planck Digital Library (submittedVersion)
- MPG.PuRe (Max Planck Society) (submittedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- tetracycline-controlled transactivator, rtTA, TetR repressor, doxycycline induction, transcriptional regulation, tetracycline resistance, mutagenesis, protein stability, synthetic promoters, HeLa cells
- MeSH terms
- Anti-Bacterial Agents, Bacterial Proteins, Carrier Proteins, Doxycycline, HeLa Cells, Humans, Mutation, Repressor Proteins, Saccharomyces cerevisiae, Tetracyclines, Trans-Activators
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