CUP2 binds in a bipartite manner to upstream activation sequence c in the promoter of the yeast copper metallothionein gene

Induction of the copper metallothionein (CUP1) gene of the yeast Saccharomyces cerevisiae is achieved by CUP2, a transcriptional activator protein which has a metal-dependent DNA-binding activity. It is thought that metal binding to CUP2 results in the formation of several looped regions of the protein which then are capable of binding to DNA. CUP2 binds to a site near the CUP1 gene called upstream activation sequence c (UASc), an imperfect inverted repeat. While CUP2 binds to both half-sites of UASc, the upstream half-site appears to be more important for transcriptional activity. A variant of CUP2 called ace1, in which cysteine-11 is replaced by tyrosine, binds to DNA but is incapable of activating transcription. We have used hydroxyl radical footprinting and missing nucleoside analysis to examine the complexes of wild-type CUP2 and the ace1 mutant protein with UASc. Our results indicate that ace1 interacts with a smaller portion of UASc than does CUP2, providing further evidence that the DNA-binding domain of CUP2 is complex, composed of two or more elements that recognize distinct features of UASc. We also show that CUP2 itself binds slightly differently to the two half-sites of UASc. While CUP2 and ace1 bind in a rather similar manner to the downstream half-site, in the upstream half-site CUP2 makes more extensive interactions. Our results suggest that the more crucial role that the upstream half-site plays in transcriptional activation may be due to differences in how CUP2 binds to each of the half-sites of UASc.