Figure 3

Evidence that ZRE sequences identified by RSAT are functional Zap1 binding sites. A) Electrophoretic mobility shift assay of candidate ZREs. Radiolabeled double-stranded oligonucleotides (0.5 pmol, 10,000 cpm) containing potential ZRE sequences from the indicated promoters were used as probes. The probes were mixed with 0 (-), 0.2 (±), 0.4 (+), or 0.8 (‡) μg per reaction of purified Zap1 DNA binding domain (Zap1_DBD). The arrow indicates the Zap1_DBD-DNA complex. The bona fide ZRE from TSA1 was used as a positive control and a mutant nonfunctional allele of that sequence (TSA1m) was used as a negative control. B) Nonrandom distribution of ZRE-like sequences in candidate Zap1 target gene promoters. In the upper panel, the positions of ZRE-like sequences in candidate Zap1 target promoters are plotted relative to the distance from the ATG start codon of the corresponding ORF. In the lower panel, the positions of ZRE-like sequences identified in the promoters of genes not showing zinc- and/or Zap1-responsive gene expression are plotted.