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. T he repressor of the Tn10 tetracycline (Tc) resistance operon of Escherichia coli (TetR) recognizes its genuine operator (tetO) with unusual specificity (1). The interaction between repressor and operator is efficiently prevented by Tc, particularly by doxycycline (Dox) that binds to TetR with high affinity (2). These parameters, as well as the fact that Dox, a nontoxic compound widely used in medicine, readily traverses cell membranes, have made the elements of the tet resistance operon attractive for the development of a transcription control system that would function in higher eukaryotic cells. It was expected that, because of their evolutionary distance, the prokaryotic regulatory elements would not interfere with the metabolism of, e.g., a mammalian cell. Accordingly, it appeared feasible to superimpose onto the complex regulatory network of a cell an independent control circuit that could be governed from outside at will. Indeed, by fusing TetR with transcription activation domains, Tc controlled transactivators (tTAs) were obtained that efficiently activate P tet , minimal promoters fused downstream of an array of tetO sequences (3, 4). The presence of Dox would prevent this activation.A TetR mutant containing four amino acid exchanges of which three are located in the protein core, where inducer is bound and triggers the conformational change necessary for induction and where dimerization takes place (5, 6), exhibits a reverse phenotype when fused to a transcription activator (C-terminal portion of VP16 of herpes simplex virus) and examined in mammalian cells (7). This mutant, called rtTA, requires Dox or anhydrotetracycline for activation of P tet . Both Dox-controlled transcription activation systems, which operate in a complementary way, have been widely used in studies of gene function in various cellular systems, as well as in whole organisms including yeast, plants, Drosophila, mice, and rats (for review see ref. 8).Despite numerous successful applications, the currently available Tet regulatory systems show some limitations. Here, we focus on the previously described rtTA, which requires Dox or anhydr...
