Transactivator mutants with altered effector specificity allow selective regulation of two genes by tetracycline variants

Engineered Tet repressors with recognition specificity for the tetO-4C5G operator variant

“…We then searched for TetR variants binding specifically to tetO-4C5G. The results described here should increase the possibilities for independent regulation of two genes in the same cell (Kamionka et al, 2004b;Krueger et al, 2004).…”

Tetracycline Derivatives: Alternative Effectors for Tet Transregulators

“…Cells were harvested 24 h after induction. Luciferase activities and protein concentrations were determined as described (9). The expression response curves are shown in Figure 2, A and B. Activation is fully abolished at effector concentrations between 1 and 100 ng/mL.…”

“…After 24 h, luciferase activity was determined as described. Values represent the means of triplicate samples with standard deviations given in ALU/g total cell protein corrected for transfection efficiency (9). The value obtained without effector was standardized to 1 corrected ALU/g protein.…”

“…Transfection experiments were performed as described previously (9). Transiently transfected rtTA2 S -M2 reacts to dox at lower concentrations than in the cell line HR2 S M2L (1 ng/mL versus 10 ng/mL), and tc leads to reasonable activation at concentrations below 1000 ng/mL.…”

Phenotypes of Combined Tet Repressor Mutants for Effector and Operator Recognition and Allostery

“…Besides shedding light on the interdependency of TetR activities and the general mechanism of protein allostery, this might yield TetR variants for tc-based multi-gene regulation systems in one cell [Krueger et al, 2004;Kamionka et al, 2004b]. We establish that operator and inducer recognition are independent properties allowing their functional combination, while TetR mutations leading to a reverse phenotype infl uence operator and inducer binding.…”