Transcriptional regulator Leu3 of Saccharomyces cerevisiae: separation of activator and repressor functions.

Abstract

The Leu3 protein of Saccharomyces cerevisiae binds to specific DNA sequences present in the 5' noncoding region of at least five RNA polymerase II-transcribed genes. Leu3 (26). Third, in the absence of a-IPM, Leu3p acts as a repressor of transcription (5,26). Leu3p thus appears to be a molecular on-off switch, where "off" means not just the absence of activation, but actual suppression of transcription below basal levels.Three major functions of the 886-residue-long Leu3p (DNA binding, transcriptional activation, and modulation by a-IPM) have been identified, and preliminary assignments for the location of regions responsible for these functions along the primary structure of the protein have been made. The DNA-binding domain is located near the N terminus where residues 37 to 67 conform to the Zn(II)2Cys6 binuclear cluster found in a number of lower eukaryotic DNA-binding proteins, including the well-studied Gal4 protein (Gal4p) (2).

Regulation of transcription in mammalian cells by yeast Leu3p and externally supplied inducer

“…To substantiate further the idea that Leu3p is modulated in a mammalian environment in a fashion similar to what had been seen in yeast, we performed in vitro transcription assays using nuclear extract from untransfected 30A5 cells and Leu3p that had been purified to near homogeneity from yeast cells overproducing the protein [26]. Extract was incubated with a template that contained the Ad2-ML promoter followed by a G-free cassette (Fig.…”

“…The antibodies used were prepared against a chimeric protein that contained the 28 Cterminal amino acid residues of Leu3p fused to key limpet hemocyanin [26]. Immunoblotting was performed following existing protocols (Amersham International Life Science).…”

Yeast Transcriptional Regulator Leu3p

“…Leu3p-In a previous paper, we initiated an analysis aimed at identifying residues of Leu3p that were important for AD masking (10). Since available evidence indicated that neither the extended DNA binding domain (DB, residues 1-173) nor a region adjacent to the AD (residues 774 -854) were required for modulation of Leu3p, whereas deletion of residues 174 -773 abolished modulation (9,11,20, and this paper), the analysis focused on the "middle region" (MR, residues 174 -773). Starting with a slow growing strain that contained a mutant Leu3p whose AD was permanently masked (Leu3p (D872N/D874N), designated Leu3-dd), faster growing suppressors were isolated following mutagenic PCR of the entire MR.…”

“…"Masking-unmasking" describes an equilibrium between inactive and active conformations of Leu3p that is influenced by ␣-IPM. "Repression" refers to a negative effect by Leu3p on transcription that requires neither the MR nor the AD of Leu3p (20). It may involve a separate negative factor (27).…”

“…The open reading frame encoding Leu3p indicates a monomer length of 886 residues (4,5). Leu3p exists as a dimer, both in the presence and absence of DNA (6,7). It recognizes the sequence 5Ј-CCGN 4 CGG-3Ј (8).…”