Nature 330, 670 - 672 (23 December 1987); doi:10.1038/330670a0

Transcription in yeast activated by a putative amphipathic helix linked to a DNA binding unit

Edward Giniger^* & Mark Ptashne

Department of Biochemistry and Molecular Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA
^*Present address: Howard Hughes Medical Institute, Department of Physiology, University of California, San Francisco, California 94143, USA.

Gene activation by a DNA-binding regulatory protein in yeast requires the protein to have two components: one to recognize a specific DNA sequence and a second, the 'activating region', to interact with a general transcription factor or perhaps with RNA polymerase^1,2. The activating regions that have been characterized are acidic^3,4, and mutational analysis of one indicates that this acidity is important for activity⁶. Here we report the design of an artificial protein bearing a novel 15-amino acid peptide linked to a DNA binding fragment of the yeast regulatory protein GAL4 (refs 7–10). The synthetic peptide is acidic and should it form an -helix, that helix would be amphipathic, having one hydrophilic face bearing the acidic residues, and one hydrophobic face¹¹. When expressed in yeast, the artificial protein bearing this peptide efficiently activates the GAL1 gene which is ordinarily activated by GAL4 (refs 12, 13). An otherwise identical protein with the novel 15 amino acids in a scrambled order, and which is thus unable to form an amphipathic structure, does not activate GAL1 transcription.

References

1.

1. Ptashne, M. Nature 322, 697-701 (1986). 2. Struhl, K. Cell 49, 295-297 (1987). 3. Hope, I. A. & Struhl, K. Cell 46, 885-894 (1986). 4. Ma, J. & Ptashne, M. Cell 48, 847-853 (1987). 5. Ma, J. & Ptashne, M. Cell 51, 113-119 (1987). 6. Gill, G. & Ptashne, M. Cell 51, 121-126 (1987). 7. Klar, A. & Halvorsen, H. Molec. gen. Genet. 135, 203-212 (1974). 8. Bram, R. & Kornberg, R. D. Proc. natn. Acad. Sci. U.S.A. 82, 43-47 (1985). 9. Giniger, E., Varnum, S. M. & Ptashne, M. Cell 40, 767-774 (1985). 10. Keegan, L., Gill, G. & Ptashne, M. Science 231, 699-704 (1986). 11. O'Neil, K. T., Wolfe, H. R. Jr, Erickson-Viitanen, S. & DeGrado, W. F. Science 236, 1454-1456 (1987). 12. Hopper, J., Broach, J. & Rowe, L. Proc. natn. Acad. Sci. U.S.A. 75, 2878-2882 (1978). 13. St. John, T. & Davis, R. /. molec. Biol. 152, 285-315 (1981). 14. West, R. Jr, Yocum, R. & Ptashne, M. Molec. cell. Biol. 4, 2467-2478 (1984). 15. Yocum, R., Hanley, S., West, R. Jr & Ptashne, M. Molec. cell. Biol. 4, 1985-1998 (1984). 16. Maniatis, T., Fritsch, E. & Sambrook, J. Molecular Cloning, a Laboratory Manual (Cold Spring Harbor, New York, 1982). 17. Ito, H., Fukuda, Y., Murata, K. K Kimura, A. J. Bact. 153, 163-168 (1983). 18. Sherman, F., Fink, G. & Hicks, J. Methods in Yeast Genetics (Cold Spring Harbor, New York, 1983). 19. Wharton, R. P. thesis, Harvard Univ. (1986). 20. Silver, P. A., Keegan, L. P. & Ptashne, M. Proc. natn. Acad. Sci. U.S.A. 81, 5951-5955 (1984). 21. Church, G. & Gilbert, W. Proc. natn. Acad. Sci. U.S.A. 81, 1991-1995 (1984). 22. Pabo, C. O. & Lewis, M. Nature 298, 443-447 (1982). 23. Hochschild, A., Irwin, N. & Ptashne, M. Cell 32, 319-325 (1983). 24. Anderson, J. A., Ptashne, M. & Harrison, S. C. Nature 316, 596-601 (1985). 25. Bushman, F. D. & Ptashne, M. Proc. natn. Acad. Sci. U.S.A. 83, 9353-9357 (1986). 26. Eisenberg, D., Schwarz, E., Komaromy, M. & Wall, R. J. molec. Biol. 179, 125-142 (1984). 27. Erickson-Viitanen, S. & DeGrado, W. F. Meth. Enzym. 139, 455-478 (1987). 28. Chothia, C., Levitt, M. & Richardson, D. J. molec. Biol. 145, 215-250 (1981).