IDENTIFICATION OF A COPPER-INDUCED INTRAMOLECULAR INTERACTION IN THE TRANSCRIPTION FACTOR MAC1 FROM SACCHAROMYCES-CEREVISIAE

Mad mediates copper (Cu)-dependent expression of genes involved in hig h-affinity uptake of copper ions in Saccharomyces cerevisiae. Mad is a transcriptional activator in Cu-deficient cells, but is inhibited in Cu-replete cells. Mad resides within the nucleus in both Cu-deficient and Cu-loaded cells. Cu inhibition of Mad appears to result from bindi ng of eight copper ions within a C-terminal segment consisting of two Cys-rich motifs. In addition, two zinc ions are bound within the N-ter minal DNA-binding domain. Only 4-5 mel. eq. Cu are bound to a mutant M ad (His279Gln substitution) that is impervious to Cu inhibition. The C uMac1 complex is luminescent, indicative of copper bound in the Cu(I) state. Cu binding induces a molecular switch resulting in an intramole cular interaction in Mad between the N-terminal DNA-binding domain and the C-terminal activation domain. This allosteric interaction is Cu d ependent and is not observed when Mad contained the mutant His279Gln s ubstitution. Fusion of the minimal DNA-binding domain of Mac1 (residue s 1-159) to the minimal Cu-binding activation domain (residues 252-341 ) yields a functional Cu-regulated transcriptional activator. These re sults suggest that Cu repression of Mac1 arises from a Cu-induced intr amolecular interaction that inhibits both DNA binding and transactivat ion activities.