The yeast transcription factor Mac1 binds to DNA in a modular fashion

Mad is a metalloregulatory protein that regulates expression of the high affinity copper transport system in the yeast Saccharomyces cerevisiae, Under conditions of high copper concentration, Mad represses transcription of genes coding for copper transport proteins. Mad binds to DNA sequences called copper response elements (CuREs), which have the consensus sequence 5'-TTTGC(T/G)C(A/G)-3'. Mad contains two zinc binding sites, a copper binding site, and the sequence motif RGRP, which has been found in other proteins to mediate binding to the minor groove of AT-rich sequences in DNA. We have used hydroxyl radical footprinting, missing nucleoside, and methylation interference experiments to investigate the structure of the complex of the DNA binding domain of Mac1 (called here Mac1(t)) with the two CuRE sites found in the yeast CTR1 promoter. We conclude from these experiments that Mac1(t) binds in a modular fashion to DNA, with its RGRP AT-hook motif interacting with the TTT sequence at the 5' end of the CTR1 CuRE site, and with another DNA-binding module(s) binding in the adjacent major groove in the GCTCA sequence.