A regulatory system for the in-depth study of gene functions in higher eukaryotic cells has been developed. It is based on the tetracycline-controlled transactivators and reverse tTA, which were remodeled to discriminate efficiently between two different promoters. The system permits one to control reversibly the activity of two genes, or two alleles of a gene, in a mutually exclusive way, and also allows one to abrogate the activities of both. This dual regulatory circuit, which can be operated by a single effector substance such as doxycycline, overcomes limitations of conventional genetic approaches. The conditional mutants that can now be generated will be useful for the study of gene function in vitro and in vivo. In addition, the system may be of value for a variety of practical applications, including gene therapy.

We describe a modification of the tetracycline-inducible eukaryotic gene expression system with decreased basal levels of expression in HeLa cells. It employs the tetracycline-inducible transactivator and a tetracycline-regulated repressor fusion acting on the same promoter. To avoid heterodimerization or competition for the same DNA site, each was provided with different DNA recognition and/or protein dimerization specificities. We achieved active silencing in the uninduced state resulting in approximately 6-fold reduced levels of basal transcription and several hundred-fold activation of gene expression upon addition of tetracycline.

Non-inducible tetracycline repressor (TetR) mutants were grouped in three structurally distinct classes. We quantitated in vivo operator binding, inducibility, and in vitro tetracycline binding of mutants from each class. Mutation of residues close to tetracycline (class 1) leads to reduced affinity for the drug. Mutation of residues located at the connection of the DNA-reading head with the protein core (class 2) and at the dimerization interface (class 3) bind inducer with the same affinity as wild-type TetR. These mutations interfere with the induced, but not the operator-binding conformation of TetR. The affinity of some class 1 mutants for tetracycline is less affected than their inducibility, suggesting that the mutated residues are important for triggering those conformational changes necessary for induction.

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We constructed 10 different variants of TetR by substituting all or some of the residues in the alpha-helix-turn-alpha-helix (HTH) operator binding motif with the respective amino acids from LacI or 434Cro. The variants were soluble, negative transdominant over tetR in vivo, and as active as wild-type TetR in tetracycline binding in vitro. The urea-induced denaturation of the 10 variants occurs in single reversible transitions, which are centered around 4.3 M urea. Denaturation is concentration-dependent, supporting a simple two-state mechanism in which the folded dimeric protein is in equilibrium with unfolded monomers. An analysis according to the two-state model yields a Gibbs free energy of stabilization (at 0 M urea, 25 degrees C) of about 75 kJ/mol, typical for dimeric proteins of this size. Even a deletion of 24 residues from the reading head decreased the stability by only 2.7 kJ/mol. These results suggest that the DNA reading head of Tet repressor is a thermodynamically independent domain and that the thermodynamic stability of the Tet repressor dimer is determined by the association of the dimerization domains of the individual monomers. Variants containing replacements in the first alpha-helix of HTH did not show any DNA binding activity whatsoever. We attribute this to the alteration of the two N-terminal residues in this alpha-helix. TetR variants were active in nonspecific DNA binding, when either all or only the solvent-exposed residues in the recognition alpha-helix of HTH were exchanged to the respective LacI sequence. Replacement of the same residues by the respective amino acids from 434Cro yielded hybrid proteins that specifically recognize tetO in vitro. Taken together, these results establish that the similarity of operator recognition between 434Cro and TetR is greater than between TetR and LacI and confirm that prediction of the recognized DNA sequence is not obvious from the sequence of the respective HTH or recognition alpha-helix.