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. 1999 Feb 2;96(3):1013–1018. doi: 10.1073/pnas.96.3.1013

Figure 3.

Figure 3

DNA-binding and dimerization specificities of various Tc-controlled transactivators. (A) (Upper) Schematic outline of the genes encoding tTA2B6C and tTA2B4C. The 4C and 6C DNA-binding domains are located N-, the transcriptional activation domain (AD) C-terminally. When exposed to operator DNA, the two transactivators discriminate between 6C (dark), 4C (light), and 6C/4C (dark/light) operator DNA. (Lower) DNA retardation experiments (for experimental detail, see ref. 15) in which the three radio-labeled tet operator DNAs were exposed to tTA2B6C and tTA2B4C, respectively, in presence (1 μg/ml) and absence of Dox. Complex formation was detected exclusively between transactivators and their cognate operator sequences in absence of Dox. Neither tTA2B6C nor tTA2B4C binds the hybrid 6C/4C operator DNA. (B) Demonstration of heterodimer formation between tTA2B6C and tTA2B4C. (Upper) Outline of the experiment in which the two transactivators were produced simultaneously in HeLa cells. The expected heterodimers bind to the hybrid 6C/4C operator DNA, as verified in the DNA retardation experiment depicted in the lower part where 6C/4C operator DNA is shifted in absence of Dox. All designations and symbols are as in A. (C) Interaction of tTA2E4C with its cognate operator DNA. (Upper) Schematic outline of the experiment as in A and B; the E-class Tet repressor is indicated by stipples. The retardation experiment (Lower) shows that tTA2E4C discriminates well between 4C and 6C operator DNA. A faint band is, however, visible also with the composite 6C/4C operator, indicating some affinity between tTA2E4C and the 6C/4C operator DNA. (D) The E- and B-type transactivators do not heterodimerize (outlined, Upper). The two transactivators tTA2E4C and tTA2B6C were produced simultaneously as in B and were exposed to the various operator DNAs. Whereas both selectively bind their cognate operators, there is no sign of heterodimerization. The faint band indicating a 6C/4C operator DNA transactivator complex is caused by the low affinity of tTA2E4C to this DNA, as seen in C.