AUTOACTIVATION BY A CANDIDA-GLABRATA COPPER METALLOREGULATORY TRANSCRIPTION FACTOR REQUIRES CRITICAL MINOR-GROOVE INTERACTIONS

Rapid transcriptional autoactivation of the Candida glabrata AMT1 copp er metalloregulatory transcription factor gene is essential for surviv al in the presence of high extracellular copper concentrations. Analys is of the interactions between purified recombinant AMT1 protein and t he AMT1 promoter metal regulatory element was carried out by a combina tion of missing-nucleoside analysis, ethylation interference, site-dir ected mutagenesis, and quantitative in vitro DNA binding studies, The results of these experiments demonstrate that monomeric AMT1 binds the metal regulatory element with very high affinity and utilizes critica l contacts in both the major and minor grooves, A single adenosine res idue in the minor groove, conserved in all known yeast Cu metalloregul atory transcription factor DNA binding sites, plays a critical role in both AMT1 DNA binding in vitro and Cu-responsive AMT1 gene transcript ion in vivo. Furthermore, a mutation in the AMT1 Cu-activated DNA bind ing domain which converts a single arginine, found in a conserved mino r groove binding domain, to lysine markedly reduces AMT1 DNA binding a ffinity in vitro and results in a severe defect in the ability of C. g labrata cells to mount a protective response against Cu toxicity.