The Tet regulatory principles: The left part of the figure outlines the mode of action of the tetracycline controlled transactivator (tTA). The upper line represents the tTA gene, driven by an appropriate promoter (Psp) and followed by a poly A site, (A)n. In the absence of the effector molecule doxycycline (Dox), tTA will bind to its cognate binding site, the tet operators tetO in the responsive promoter (Ptet) shaded in grey, and activate transcription of the gene under study (x). The presence of Dox will prevent tTA from binding thus abolishing gene expression, hence the term "Tet Off" system. The left lower part of the figure shows a dose-response analysis of doxycycline control over tTA activity. Gene expression is maximal in the absence of the effector molecule but increasing effector concentrations lead to a gradual decrease of transcriptional activation to reach background levels at Dox concentrations >= 2 ng/ml.
The right part of the figure outlines the reverse (rtTA dependent) Tet System. rtTA differs from tTA by 4 amino acid substitutions in the TetR moiety leading to a reversal of function with respect to the effector substance: rtTA requires tetracyclines like Dox for binding to tetO and therefore will activate transcription of Ptet only in the presence of the effector (hence the term "Tet-On" system). The dose response curve on the lower right part of the figure outlines the effect of Dox on rtTA mediated transcriptional activation (dotted line). Increase of effector concentrations above 20 ng/ml will lead to a gradual increase of rtTA dependent gene expression with maximal levels being reached at about 1 µg/ml. rtTA2-syn1 (solid line) the synthetic, improved reverse transactivator shows higher sensitivity towards Dox when compared to rtTA. in this case, full activation is achieved at about 80 ng/ml thus at 10-fold lower effector concentrations when compared to rtTA. This has important implications mainly in transgenic animals where high doxycycline concentrations are difficult to reach in certain tissues/organs (e.g. in the brain).
Figure 2 - Structure of Tet-responsive promoters
Tet-responsive promoters are fusions between heptamerized tetO sequences (indicated as grey squares) and minimal promoters derived from viral or cellular RNA polymerase II promoters. The promoters shown: Ptet-1; Ptet-14 and Ptetbi-1 are all derivatives of the human CMV promoter. The originally published Tet-responsive promoter PhCMV*-1 (now named Ptet-1) comprises the sequence between -53 to +75 of PhCMV with the heptamerized tet operators inserted at position -53. Positions spanning promoter regions and tet operator sequences are indicated with respect to the start site of transcription (+1).
Ptet-14 differs from Ptet-1 through reduction of the CMV promoter part from +75 to +12. In addition, the spacers separating the tet operators were optimized e.g. by removing potential eukaryotic transcription factor binding sites. In the bidirectional promoter Ptetbi-1 shown at the bottom of the figure the heptamerized tetO sequences are flanked by two divergently oriented CMV derived minimal promoters. The drawings are not to scale.
Figure 3 - Tetracycline-controlled fusion proteins
The figure represents fusions between TetR or rTetR (light green) and transcription activating (dark green) or repressing (orange) domains. tTA, the originally described transactivator consists of 207 amino acids derived from TetR and the C-terminal 130 amino acids of HSV protein VP16 . In tTA2, the VP16 part was reduced to three consecutive minimal activation domains (F), each consisting of 13 amino acids. tTA2-syn , the amino acid sequence of which is identical to tTA2 is encoded by a synthetic gene which was optimized for expression in mammalian cells.
The originally described reverse transactivator rtTA differs from tTA by four amino acid exchanges in the TetR moiety (indicated as asterisks) .
rtTA2-syn1 contains a different set of mutations when compared to rtTA. They mediate the reverse phenotype with increased doxycycline responsiveness (see Figure 1). Like tTAsyn, rtTA2-syn1 is based on an optimized open reading frame design including the three minimal activating domains.
The transcriptional repressor tTS Kid is a fusion between TetR and a 62 amino acid long repression moiety comprising the KRAB domain of human kidney protein Kid-1. This domain is connected to TetR via a nuclear localisation signal (n).
Figure 4 - Shielding and activation of minimal promoters
Depending on the site of integration in the chromosome minimal promoters can be subject to activation by nearby enhancers (enh). In case of Ptet this will lead to expression of the gene under study (x) independent of a Tet transactivator. By binding to the tetO sequences within Ptet, tTS the tetracycline controlled transcriptional silencer can protect Pmin from outside activation. As tTS is TetR derived it will bind to tetO only in the absence of doxycycline; addition of the effector substance will lead to dissociation of tTS and activation of gene expression via any compatible reverse transactivator.