Tetracycline-regulated promoters

Scientific aspects

Transposons are mobile genetic elements which can insert at random into plasmids or a bacterial chromosome independently of the host cell recombination system.  Transposons carry genes that confer new phenotypes on the host cell such as antibiotic resistance.

The Tetracycline resistance operon is carried by the Escherichia coli transposon (Tn) 10. This operon has a negative mode of operation. The interaction between a repressor protein encoded by the operon,TetR, and a DNA sequence to which it binds, the tet operator (tetO), represses the activity of a promoter placed near the operator. In the absence of an inducer,TetR binds to tetO and prevents transcription.  Transcription can be turned on when an inducer, such as tetracycline, binds to TetR and causes a conformation change that prevents TetR from remaining bound to the operator. When the operator site is not bound, the activity of the promoter is restored.

In plants, the system has been adapted in two ways

• as a promoter repressing system, basically using the system as described, and
• as a promoter activating system, where TetR is modified to activate gene transcription, instead of inhibiting transcription.

In one system, a CaMV 35S promoter is modified by introducing a tet operator sequence upstream and downstream of the TATA box. In the absence of tetracycline, overexpressed TetR binds to the tet operator and prevents gene expression. In the presence of tetracycline, TetR no longer binds the operator and gene expression is turned on.

In tobacco, the expression of the tetracycline-inducible CaMV promoter could be modulated up to 500-fold. This inducible promoter has also worked in tomato and potato.

Unfortunately, the system presents some problems. For tetracycline to work as an inducer, it must be supplied continuously to the medium due in part to the short-half life of the antibiotic.  In addition, TetR must be in high concentration to be effective as a repressor as it has to compete with at least forty proteins that assemble around the TATA box. For some plants, such as Arabidopsis, high concentrations of the repressor are toxic and alter the photosynthetic physiology of the plant.

Due to the drawbacks of the promoter repressing system, TetR has also been converted to an activator of gene expression. In one version of a promoter activating system, TetR is fused to the acidic activation sequence of the herpes simplex virus protein 16 (VP16), forming a tetracycline transactivator (tTA) fusion protein which is has the DNA binding specificity of TetR and the promoter activating function of VP16. In the absence of tetracycline,  tTA binds to tet operator sequences placed upstream of a TATA box in a target promoter and activates transcription. When tetracycline is provided, it forms a complex with tTA and releases the operator, thus, turning off gene transcription.

In contrast to the wild type TetR, tTA does not need to compete with endogenous transcription factors for binding sites. The system has worked in tobacco and in Arabidopsis.

Despite the advantages of the Tet activating system, the plants must be in the presence of tetracycline to turn transcription off. That implies a continuous supply of the antibiotic. Also the original promoter that contains the tet operators is prone to silencing over time. Work has been done to improve the efficacy of the promoter.

The information contained in this page was believed to be correct at the time it was collated. New patents and patent applications, altered status of patents, and case law may have resulted in changes in the landscape. CAMBIA makes no warranty that it is correct or up to date at this time and accepts no liability for any use that might be made of it. Corrections or updates to the information are welcome. Please send an email to info@bios.net.

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