Autoactivation by a Candida glabrata copper metalloregulatory transcription factor requires critical minor groove interactions.
Rapid transcriptional autoactivation of the Candida glabrata AMT1 copper metalloregulatory transcription factor gene is essential for survival in the presence of high extracellular copper concentrations. Analysis of the interactions between purified recombinant AMT1 protein and the AMT1 promoter metal regulatory element was carried out by a combination of missing-nucleoside analysis, ethylation interference, site-directed 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 critical contacts in both the major and minor grooves. A single adenosine residue in the minor groove, conserved in all known yeast Cu metalloregulatory transcription factor DNA binding sites, plays a critical role in both AMT1 DNA binding in vitro and Cu-responsive AMT1 gene transcription in vivo. Furthermore, a mutation in the AMT1 Cu-activated DNA binding domain which converts a single arginine, found in a conserved minor groove binding domain, to lysine markedly reduces AMT1 DNA binding affinity in vitro and results in a severe defect in the ability of C. glabrata cells to mount a protective response against Cu toxicity.
Koch
K A
KA
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.
Thiele
D J
DJ
D001483:Base Sequence; D001665:Binding Sites; D002175:Candida / Q000187:drug effects / Q000235:genetics*; D017124:Conserved Sequence; D003300:Copper / Q000494:pharmacology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins; D015966:Gene Expression Regulation, Fungal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D014157:Transcription Factors; C069807:AMT1 protein, Candida glabrate; D003300:Copper