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

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. 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...

Doxycycline-regulated lentiviral vector system with a novel reverse transactivator rtTA2S-M2 shows a tight control of gene expression in vitro and in vivo

“…Recently, two reports on the successful use of novel mutated versions of the tetracycline-dependent transcriptional activators 6 have been published. Vigna et al 30 used Tet-off application with a tetracycline transactivator …”

“…The sections were stained with X-gal for b-galactosidase activity. The X-gal reagent (5-bromo-4-chloro-3-indolylb-D-galactoside) (MBI Fermentas, Lithuania) was diluted 1:100 with X-gal solution (5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , 2 mM MgCl 2 ) added on the sections and incubated for 16 h at 371C. Sections were rinsed with phosphate-buffered saline and counterstained with Mayer's Carmalum.…”

“…Also, relatively inefficient inducibility by dox via the rtTA in some organs, eg brain, and instability of the rtTA have limited the use of the Tet-on system. 5 The recently described mutated reverse transactivator rtTA2 S -M2 6 has highly improved properties with respect to specificity, stability and inducibility. It functions at 10-fold lower dox concentration than the original rtTA, is more stable in eukaryotic cells and most importantly, causes no background expression in the absence of dox.…”

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR

“…7,8 Activation of the tet-regulated promoter by dox is dose-dependent and transgene expression can be controlled over a narrow window of dox concentrations. 9 Another advantage of this system, particularly for cloning into rAAV which has a limited insert size of approximately 4.5 kb, is that the genes that encode the tet repressor and inducible promoter are small compared to the elements required for other systems. 10 The tet inducible system consists of two components, the tet-controlled transactivator protein (tTA), and the tet-regulated element (TRE).…”

Application of a Reversible Immortalization System for the Generation of Proliferation-Controlled Cell Lines

“…Schematic representation of the vectors used. (a) The plasmid vector pLTRGFPN contains a bi-directional tetracycline dependent promoter PbitTA that drives the expression of two divergent mRNAs encoding the TAg and the reverse transactivator rtTA2M2, respectively (Urlinger et al 2000), and a fusion protein of eGFP and neo (Colbe`re-Garapin et al 1981). The individual components of pLTRGFPN are derived from the following constructs: the bi-directional doxycycline regulated promoter P bitTA stems from pBI-I (Baron et al 1995), the SV40 TAg is followed by SV40 p(A) polyadenylation signal sequences; the rtTA2M2 sequences are derived from pUHrt62-1 (Urlinger et al 2000) an encephalomyocarditis virus internal ribosomal entry site is derived from pEMCVhyg (Verhoeyen et al 2001); the reading frame encoding an enhanced green fluorescent protein was fused at the C-terminus without the stop codon in frame to the N-terminus of the selection marker gene neo followed by a SV40 polyadenylation signal.…”