The minimal transactivation region of Saccharomyces cerevisiae Gln3p is localized to 13 amino acids.
Regulated nitrogen catabolic gene transcription in Saccharomyces cerevisiae is mediated by four positive (Gln3p and Gat1p/Nil1p) and negative (Dal80p/Uga43p and Deh1p/Nil2p/GZF3p) regulators which function in opposition to one another. All four proteins contain GATA-type zinc finger domains, and three of them (Gln3p, Dal80p, and Deh1p) have been shown to bind to GATA sequences situated upstream of genes whose expression is sensitive to nitrogen catabolite repression (NCR). The positive regulators, Gln3p and Gat1p, are able to support transcriptional activation when tethered by LexAp to the promoter of a reporter gene whose upstream activation sequences have been replaced with one or more lexA operator sites. Existing data suggest that these four proteins regulate transcription by competing with one another for binding to the GATA sequences which mediate NCR-sensitive gene expression. We show that the minimal Gln3p domain mediating transcriptional activation consists of 13 amino acids with a predicted propensity to form an alpha-helix. Genetic analysis of this region (Gln3p residues 126 to 138, QQNGEIAQLWDFN) demonstrated that alanine may be substituted for the aromatic and acidic amino acids without destroying transcriptional activation potential. Similar substitution of alanine for the two hydrophobic amino acids, isoleucine and leucine, however, destroys activation, as does introduction of basic amino acids in place of the acidic residues or introduction of proline into the center of the sequence. A point mutation in the Gln3p activation region destroys its in vivo ability to support NCR-sensitive DAL5 expression. We find no convincing evidence that NCR regulates Gln3p function by modulating the functioning of its activation region.
Svetlov
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Department of Microbiology and Immunology, University of Tennessee, Memphis 38163, USA.
Cooper
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D000595:Amino Acid Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D012097:Repressor Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D015533:Transcriptional Activation; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C071664:GLN3 protein, S cerevisiae; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors