TRANSCRIPTIONAL REGULATOR LEU3 OF SACCHAROMYCES-CEREVISIAE - SEPARATION OF ACTIVATOR AND REPRESSOR FUNCTIONS

The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA seq uences present in the 5' noncoding region of at least five RNA polymer ase II-transcribed genes. Leu3 functions as a transcriptional activato r only when the metabolic intermediate alpha-isopropylmalate is also p resent. In the absence of alpha-isopropylmalate, Leu3 causes transcrip tion to be repressed below basal levels. We show here that different p ortions of the Leu3 protein are responsible for activation and repress ion. Fusion of the 30 C-terminal residues of Leu3 to the DNA-binding d omain of the Gal4 protein created a strong cross-species activator, de monstrating that the short C-terminal region is not only required but also sufficient for transcriptional activation. Using a recently devel oped Leu3-responsive in vitro transcription assay as a test system for repression (J. Sze, M. Woontner, J. Jaehning, and G. B. Kohlhaw, Scie nce 258:1143-1145, 1992), we show that mutant forms of the Leu3 protei n that lack the activation domain still function as repressors. The sh ortest repressor thus identified had only about 15% of the mass of the full-length Leu3 protein and was centered on the DNA-binding region o f Leu3. Implications of this finding for the mechanism of repression a re discussed.