A single amino acid change in CUP2 alters its mode of DNA binding.
CUP2 is a copper-dependent transcriptional activator of the yeast CUP1 metallothionein gene. In the presence of Cu+ and Ag+) ions its DNA-binding domain is thought to fold as a cysteine-coordinated Cu cluster which recognizes the palindromic CUP1 upstream activation sequence (UASc). Using mobility shift, methylation interference, and DNase I and hydroxyl radical footprinting assays, we examined the interaction of wild-type and variant CUP2 proteins produced in Escherichia coli with the UASc. Our results suggest that CUP2 has a complex Cu-coordinated DNA-binding domain containing different parts that function as DNA-binding elements recognizing distinct sequence motifs embedded within the UASc. A single-amino-acid substitution of cysteine 11 with a tyrosine results in decreased Cu binding, apparent inactivation of one of the DNA-binding elements and a dramatic change in the recognition properties of CUP2. This variant protein interacts with only one part of the wild-type site and prefers to bind to a different half-site from the wild-type protein. Although the variant has about 10% of wild-type DNA-binding activity, it appears to be completely incapable of activating transcription.
Buchman
C
C
Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla 92093.
Skroch
P
P
Dixon
W
W
Tullius
T D
TD
Karin
M
M
D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D003300:Copper / Q000494:pharmacology*; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D003850:Deoxyribonuclease I; D005609:Free Radicals; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006878:Hydroxides; D017665:Hydroxyl Radical; D008668:Metallothionein / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic
C060256:CUP2 protein, S cerevisiae; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005609:Free Radicals; D005656:Fungal Proteins; D006878:Hydroxides; D015345:Oligonucleotide Probes; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D017665:Hydroxyl Radical; D003300:Copper; D008668:Metallothionein; D003850:Deoxyribonuclease I