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Abstract
ALCR is the specific activator of the Aspergillus nidulans ethanol-utilization pathway, mediating the induction of its own transcription and that of the structural genes alcA and aldA, encoding respectively, alcohol dehydrogenase I and aldehyde dehydrogenase. ALCR is a DNA binding protein in which 6 cysteines are coordinated in a zinc binuclear cluster. This domain was fused to glutathione-S-transferase (GST) and isolated as a GST-ALCR(7-58*) fusion protein from Escherichia coli. Mobility shift assays showed that the ALCR fusion protein binds at sites upstream of the alcA promoter. DNaseI protection footprinting experiments revealed three specific binding sites, two that are direct repeats and one that is an inverted repeat with the same half-site 5'-CCGCA-3'. The half-sites are separated by a variable number of nucleotides in both types of target. The interaction of the ALCR fusion protein with direct and inverted repeats were examined by using interference and protection footprinting assays. In both binding sites, modification of the guanines in the half-sites interfered with the formation of the DNA complex, but the adjacent ones did not. Our results suggest that the ALCR protein makes contact in the major groove of the DNA helix of the half-sites. The functionality of two out of three binding sites of the GST-ALCR protein was demonstrated after their deletion. Therefore, the region encompassing these binding sites is a cis-acting element involved in the full induction of the alcA gene.
Highlights
ALCR is the specific activator of the Aspergillus see Felenbok, 1991)
In GAL4 and LACS, it was shown that these fusion protein with direct and inverted repeats were structurescontained two Zn(I1)atoms able to form a binuclear examined by using interference and protection foot- complex of a cloverleaf type
The ALCR fusion protein bound to two restriction fragments and one PCR product of the &A promoter, each one producing a single band in the gel-shift assay (shown in Fig. 1C forthe PCR fragment)
Summary
Strains-The A. nidulans strains used were wildtype (biAl); (yA2 pantoBlO0 amdS320 amdZl8 amdA7alcR125); The HindIII-EcoRI a&A promoter fragment was ligated to a plasmid, pUC12, containing therest of the alcA coding region (Gwynne et al, 1987). The argB gene (Upshall et al, 1986) from pMA2 plasmid was integrated at thXe baI site of the &pUC12 plasmids. These constructions were used to transform an A. nidulans alc-. DNA-binding assays and thenondenaturatingpolyacrylamideelectrophoresis were performed as described previously (Kulmburg et al., 1992)
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