Abstract
BackgroundThe Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application.ResultsWe recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used.ConclusionWe developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used.
Highlights
Introduction of the mutations in bothTet repressor (TetR) parts resulted in the most active single-chain reverse tTAs (rtTAs) (sc-rtTA) variants (Fig. 3C)
We have used viral evolution to optimize the function of the Tet-On system, and identified several amino acid substitutions in the rtTA protein that greatly enhance its transcriptional activity and dox-sensitivity [24,26] (Fig. 1A)
These results demonstrate that mutations beneficial for rtTA do not necessarily improve tTA activity
Summary
Introduction of the mutations in bothTetR parts resulted in the most active sc-rtTA variants (Fig. 3C). The variant carrying the F67S and F86Y mutations in both TetR moieties requires only 16 ng/ml dox to reach this 100% activity level, which reflects a 62-fold higher dox-sensitivity than the wild-type sc-rtTA. The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). TTA variants have been isolated that carry amino acid substitutions in the TetR part and exhibit a reverse phenotype [5,6] (Fig. 1) These reverse tTAs (rtTAs) activate gene expression from tetOcontaining minimal promoters only in the presence of dox (Tet-On system). The Tn10-derived TetR moiety of tTS was replaced with other natural TetR variants to prevent heterodimerization with rtTA [8,9,10,11]
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