The p65 domain from NF-kappaB is an efficient human activator in the tetracycline-regulatable gene expression system.

The tc-responsive TetR protein allows the investigation of various transcriptional activators in respective fusion proteins. We have fused eight well-known human activator domains to the C-terminus of TetR and determined the properties of the resulting transactivators using a tetracycline-responsive promoter in three human cell lines (HeLa, BJAB, and Jurkat). Several-hundred-fold activation was exclusively obtained with the acidic p65 domain from NF-kappaB and with VP16, which served as a positive control.

Domain motions accompanying Tet repressor induction defined by changes of interspin distances at selectively labeled sites.

To investigate internal movements in Tet repressor (TetR) during induction by tetracycline (tc) we determined the interspin distances between pairs of nitroxide spin labels attached to specific sites by electron paramagnetic resonance (EPR) spectroscopy. For this purpose, we constructed six TetR variants with engineered cysteine pairs located in regions with presumed conformational changes. These are I22C and N47C in the DNA reading head, T152C/Q175C, A161C/Q175C and R128C/D180C near the tc-binding pocket, and T202C in the dimerization surface.

Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes.

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.

Tetracycline-inducible expression systems with reduced basal activity in mammalian cells.

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.

Stepwise selection of TetR variants recognizing tet operator 6C with high affinity and specificity.

The exchange of Trp43 to Arg in the sixth position of the TetR recognition alpha-helix leads to a new DNA recognition specificity for tetO-6C, however, it is bound with only low affinity. Specificity and affinity of this mutant were substantially increased by additional amino acid exchanges in the last positions of the recognition alpha-helix and the turn, which most likely play structural roles in the formation of the TetR-tetO complex. The last residue in the turn of the alpha-helix-turn-alpha-helix motif is a discriminator of binding to other tetO variants and contributes efficiently to the affinity for the newly recognized tetO-6C sequence.