Evidence for (Mac1p)2.DNA ternary complex formation in Mac1p-dependent transactivation at the CTR1 promoter

Abstract

The Mac1 protein in Saccharomyces cerevisiae is required for the expression CTR1 and FRE1, which, respectively, encode the copper permease and metal reductase that participate in copper uptake. Mac1p binds to a core GCTC sequence present as a repeated unit in the promoters of both genes. We show here that Mac1p DNA binding required an intact N-terminal protein domain that includes a likely zinc finger motif. This binding was enhanced by the presence of a TATTT sequence immediately 5' to the core GCTC, in contrast to a TTTTT one. This increased binding was demonstrated clearly in vitro in electrophoretic mobility shift assays that showed Mac1p.DNA complex formation to a single TATTTGCTC element but not to a TTTTTGCTC one. Furthermore, the fraction of Mac1p in a ternary (Mac1p)2.DNA complex in comparison to a binary Mac1p.DNA complex increased when the DNA included two TATTTGCTC elements. A similar increase in ternary complex formation was demonstrated upon homologous mutation of the FRE1 Mac1p-dependent promoter element. The in vivo importance of this ternary complex formation at the CTR1 promoter was indicated by the stronger trans-activity of this promoter mutated to contain two TATTT elements and the attenuated activity of a mutant promoter containing two TTTTT elements that in vitro supported only a weak ternary complex signal in the shift assay. The stronger binding to TATTT appeared due to a more favorable protein contact with adenine in comparison to thymine at this position. An in vivo two-hybrid analysis demonstrated a Mac1p-Mac1p protein-protein interaction. This Mac1p-Mac1p interaction may promote (Mac1p)2.DNA ternary complex formation at Mac1p-responsive upstream activating sequences.