Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity

Parts used

Key findings

• Urlinger et al. engineered novel reverse tetracycline transactivators (rtTAs) via random mutagenesis of tTA DNA and selection in Saccharomyces cerevisiae. Five new rtTA variants were identified, two showing greatly improved properties over the original rtTA, including reduced basal activity, increased doxycycline sensitivity, and enhanced stability in eukaryotic cells.
• The most improved variant, rtTA2S-M2, activated Ptet promoters at a 10-fold lower doxycycline concentration than the original rtTA, showed increased stability in eukaryotic cells, and produced no background expression in the absence of doxycycline. These improvements directly addressed limitations that had restricted rtTA use in transgenic animals.
• Synthetic codon-optimized genes encoding tTA2, rtTA2-S2, and rtTA2-M2 were designed for optimal expression in human cells. The resulting transactivators enabled stringent regulation of target genes over 4 to 5 orders of magnitude in stably transfected HeLa cells, combining tight expression control with a broad dynamic range.

Abstract

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.