Relationship between zinc content and DNA-binding activity of the DNA-binding motif of the transcription factor ALCR in Aspergillus nidulans

Abstract

The transcription factor ALCR of the ethanol utilisation pathway in Aspergillus nidulans contains a zinc binuclear motif (CysX2CysX6CysX16CysX2CysX6Cys), within the DNA-binding domain located in the N-terminal region of the ALCR protein. Specific targets have been localised in the promoter of the alcR gene, involved in the autoregulation process, and in the promoter of the structural gene alcA (encoding alcohol dehydrogenase I), which is also under the control of ALCR. The DNA-binding domain has been expressed in-Escherichia coli as a GST-ALCR (7-58*) fusion protein and also obtained as an ALCR (7-58*) peptide. Both the ALCR fusion protein and the ALCR peptide are able to bind 65Zn(II) in vitro, if reduction of cysteines occurs prior to the addition of zinc. Competition experiments showed that Cd(II), Co(II) and Cu(II) are efficient competitors for the zinc binding sites. The ALCR DNA-binding domain was shown to contain 2 mol of tightly bound Zn(II) per mole of fusion protein. Removal of the intrinsic Zn(II) requires treatment with Chelex. This treatment abolishes the ability of the protein to bind to the targets of ALCR located in the alcA and alcR promoters. The apo-ALCR DNA-binding motif could be reconstituted with Zn(II) or Cd(II), restoring specific DNA binding to both types of targets. Thus a direct relationship was shown to exist between the zinc content of ALCR and its DNA-binding activity.