Manipulation of the 'zinc cluster' region of transcriptional activator LEU3 by site-directed mutagenesis.
The transcriptional activator LEU3 of Saccharomyces cerevisiae belongs to a family of lower eukaryotic DNA binding proteins with a well-conserved DNA binding motif known as the Zn(II)2Cys6 binuclear cluster. We have constructed mutations in LEU3 that affect either one of the conserved cysteines (Cys47) or one of several amino acids located within a variable subregion of the DNA binding motif. LEU3 proteins with a mutation at Cys47 were very poor activators which could not be rescued by supplying Zn(II) to the growth medium. Mutations within the variable subregion were generally well-tolerated. Only two of seven mutations in this region generated poor activators, and both could be reactivated by Zn(II) supplements. Three of the other five mutations gave rise to activators that were better than wild type. One of these, His50Cys, exhibited a 1.5 fold increase in in vivo target gene activation and a notable increase in the affinity for target DNA. The properties of the His50Cys mutant are discussed in terms of a variant structure of the DNA binding motif. During the course of this work, evidence was obtained suggesting that only one of the two LEU3 protein-DNA complexes routinely seen actually activates transcription. The other (which may contain an additional protein factor) does not.
Bai
Y L
YL
Department of Biochemistry, Purdue University, West Lafayette, IN 47907.
Kohlhaw
G B
GB
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D015533:Transcriptional Activation; D014170:Transformation, Genetic / Q000235:genetics; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C063667:LEU3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D001616:beta-Galactosidase; D015032:Zinc; D003545:Cysteine