Functional independence of the two cysteine-rich activation domains in theyeast Mac1 transcription factor

Mac1 is a transcriptional activator whose activity is inhibited by copper i ons. Mutagenesis studies were carried out to map residues important in the copper inhibition of Mac1 activity. Seven new missense mutations were ident ified that resulted in copper-independent Mac1 transcriptional activation. All seven mutations were clustered in one of two C-terminal cysteine-rich m otifs, designated the C1 motif. All but one of the constitutive Mac1 mutati ons occurred in one of the conserved six residues in the (CXC)-C-264(X)(4)C XC(X)(2)C(X)(2)H-279 C1 motif. The lone exception was a L260S substitution. Two additional MAC1 mutations exhibiting constitutive activity were in-fra me deletions encompassing portions C1, Engineered mutations in the second c ysteine-rich motif did not yield a constitutively active Mac1. These result s are consistent with the C1 motif being the copper-regulatory switch. Both cysteine-rich motifs exhibited transactivation activity, although the C1 a ctivator was weak relative to the C2 activator. Limited copper metalloregul ation of Mac1 was observed with only the C1 activator fused to the N-termin al DNA binding domain. Thus, the two Cys-rich motifs appear to function ind ependently. The C1 motif appears to be a functional copper-regulatory domai n.