EXPERIMENTAL FIGURE 9-27 Deletion mutants of the GAL4 gene in yeast with a UAS_GAL reporter-gene construct demonstrate the separate functional domains in a transcription activator. (a) Diagram of DNA construct containing a lacZ reporter gene (encoding β-galactosidase) and TATA box ligated to UAS_GAL, a regulatory element that contains several Gal4-binding sites. The reporter-gene construct and DNA encoding wild-type or mutant (deleted) Gal4 were simultaneously introduced into mutant (gal4) yeast cells, and the activity of β-galactosidase expressed from lacZ was assayed. Activity should be high if the introduced GAL4 DNA encodes a functional protein. (b) Schematic diagrams of wild-type Gal4 and various mutant forms. Small numbers refer to positions in the wild-type sequence. Deletion of 50 amino acids from the N-terminal end destroyed the ability of Gal4 to bind to UAS_GAL and to stimulate expression of β-galactosidase from the reporter gene. Proteins with extensive deletions from the C-terminal end still bound to UAS_GAL. These results localize the DNA-binding domain to the N-terminal end of Gal4. The ability to activate β-galactosidase expression was not entirely eliminated unless somewhere between 126 and 189 or more amino acids were deleted from the C-terminal end. Thus the activation domain lies in the C-terminal region of Gal4. Proteins with internal deletions (bottom) were also able to stimulate expression of β-galactosidase, indicating that the central region of Gal4 is not crucial for its function in this assay. See J. Ma and M. Ptashne, 1987, Cell 48:847; I. A. Hope and K. Struhl, 1986, Cell 46:885; and R. Brent and M. Ptashne, 1985, Cell 43:729.