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

Regulatory elements that control tetracycline resistance in Escherichia col i were previously converted into highly specific transcription regulation s ystems that function in a wide variety of eukaryotic cells. One tetracyclin e repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled tran sactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P-tet promoters. Despite the intriguing propert ies of rtTA, its use was limited, particularly in transgenic animals, becau se of its relatively inefficient inducibility by doxycycline in some organs , its instability, and its residual affinity to tetO in absence of Dox, lea ding to elevated background activities of the target promoter. To remove th ese limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox s ensitivity. Five new rtTAs were identified, of which two have greatly impro ved properties. The most promising new transactivator, rtTA2(S)-M2, functio ns at a 10-fold lower Dox concentration than rtTA, is more stable in eukary otic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activatio n 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. The se rtTA versions combine tightness of expression control with a broad regul atory range, as previously shown for the widely applied tTA.