Manipulation of the ‘Zinc cluster’ region of transcriptional activatorLEU3be site-directed mutagenesis

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Abstract
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.