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Binding to UAS

P-galactosidase activity

▲ EXPERIMENTAL FIGURE 11-17 Deletion mutants of the GAL4 gene in yeast with a UASg^i reporter-gene construct demonstrate the separate functional domains in an activator.

(a) Diagram of DNA construct containing a lacZ reporter gene and TATA box ligated to UASgal, 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 p-galactosidase expressed from lacZ was assayed. Activity will 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 UASgal and to stimulate expression of p-galactosidase from the reporter gene. Proteins with extensive deletions from the C-terminal end still bound to UASgal. These results localize the DNA-binding domain to the N-terminal end of GAL4. The ability to activate p-galactosidase expression was not entirely eliminated unless somewhere between 126-189 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) also were able to stimulate expression of p-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.]

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