Teton Tetoff System

As we have discussed in Chapter 1, the control of transcriptional initiation is fundamentally different between eukaryotes and prokaryotes. An activator from prokaryotes is unable to bring about a transcriptional response in eukaryotes and vice versa. DNA binding is, however, species independent. The tightly regulated DNA binding properties of prokaryotic activators can be used to direct eukaryotic activation domains to drive the expression of target genes. One such system exploits the DNA properties of the E. coli tetracycline repressor.

The E. coli tet operon was originally identified as a transposon (Tn10) that confers resistance to the antibiotic tetracycline (Foster et al., 1981). The TetR protein, in a similar fashion to the lac repressor protein (LacI) we have already discussed (Chapter 1), binds to the operator of the tetracycline-resistance operon and prevents RNA polymerase from initiating transcription. Activation of the tetracycline-resistance operon occurs when tetracycline itself binds to the repressor and induces a conformational change that inhibits its DNA binding activity. The TetR protein has a very high affinity for the antibiotic (association constant ~3 x 10-9 M-1) and will dissociate from its DNA binding site when tetracycline is present at low concentrations (Takahashi, Degenkolb and Hillen, 1991). The regulated DNA binding activity of TetR cannot itself elicit a transcriptional response in eukaryotes, but can if the protein is fused to a eukaryotic transcriptional activator domain. The use of the tet system to drive target gene expression in eukaryotes relies on the insertion of two recombinant DNA molecules into the host cell (Figure 8.7).

• Regulator plasmid - produces a version of the E. coli tetracycline repressor (TetR) that is fused to the transcriptional activation domain of the herpes simplex virus VP16 protein. The fusion protein is constitutively produced in the host cell from the CMV promoter.

• Response plasmid - contains the target gene cloned downstream of mul-timerised copies of the tetracycline operator (tetO) DNA sequence that form a tetracycline response element (TRE) cloned into a minimal CMV promoter that is not, on its own, able to support gene activation.

In the absence of tetracycline, the TetR-VP16 fusion protein will bind to the TRE and activate transcription of the target gene. Upon the addition of tetracycline to the cells, however, TetR will dissociate and target gene transcription will be turned off (Gossen and Bujard, 1992). That is, the addition of tetracycline turns target gene expression off. The use of the tet system has become more prevalent due to the existence of a mutant version of TetR. The mutant tetracycline repressor contains four amino acid changes (E71K, D95N, L101S and G102D) from the wild-type protein that radically alter its DNA binding properties. Rather than tetracycline inhibiting its DNA binding properties, the mutant protein, called rTetR for reverse tetracycline repressor, will only bind DNA in the presence of tetracycline (Gossen et al., 1995). This means that, with the appropriate TetR fusion to the activation domain of VP16, target gene expression can either be inhibited or activated in the presence of tetracycline.

• Tet-off uses the wild-type TetR protein fused to VP16. Target gene expression is active in the absence of tetracycline but not in its presence.

Tet-Off

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