No abstract

The ALCR protein is the transcriptional activator of the ethanol utilization pathway in the filamentous fungus AspergiUlus nidulans. This activator belongs to a family of fungal proteins having a conserved DNA-binding domain containing six cysteines (C6 class) with some striking features. At variance with other motifs of this class, the binding domain of ALCR is strongly asymmetrical in relation to the central cysteines and moreover was predicted to adopt a helix-turn-helix structure. This domain of ALCR was synthesized in Escherichia coli and purified as a glutathione-S-transferase fusion protein. Our results show that the transcriptional activator ALCR is a DNA-binding protein. The DNA-binding motif contains zinc that is necessary for the specific DNA binding. The ALCR peptide binds upstream of the coding region of alcR to two specific targets with different affinities that are characterized by a conserved 5-nucleotide core, 5'-CCGCA-3' (or its reverse). One site, the lower-affinity binding site, is a direct repeat, and the other, the higher-affinity binding site, is a palindromic sequence with dyad symmetry. Therefore, the ALCR binding protein is able to recognize one DNA sequence in two different configurations. An alcR mutant obtained by deletion of the two specific targets in the cis-acting region of the alcR gene is unable to grow on ethanol and does not express any alcohol dehydrogenase activity. These results demonstrate that the binding sites are in vivo functional targets (UASaic) for the ALCR protein in A. nidulans. They corroborate prior evidence that alcR is autoregulated.Positive control mechanisms in eukaryotes have been extensively characterized. They are mediated by transcription factors that bind to specific DNA targets. The expression of genes encoding the ethanol utilization enzymes in the ascomycete Aspergillus nidulans is regulated by the pathway-specific transactivator ALCR. In conditions of induction (by ethanol or gratuitous inducers like ethylmethylketone), the ALCR protein is necessary for the expression of the two structural genes alcA, encoding alcohol dehydrogenase I, and aldA, encoding aldehyde dehydrogenase (30, 36). Transcription of these two genes can be strongly induced, and this property was widely used for the expression of heterologous proteins (for a review, see reference 11). The expression of the alcR gene is inducible, positively autoregulated, and subjected to carbon catabolite repression under the control of the negatively acting gene creA (11,25,30). The three genes of the ethanol regulon were cloned and sequenced (12,16,26,32), and the creA gene identified by Bailey and Arst (2) was also cloned (8) and sequenced (9). The transcription factor ALCR is 821 amino acids long (12) and contains a sequence of six Cys residues, Cys-X2-Cys-X6-Cys-X16-Cys-X2-Cys-X6-Cys within its N-terminal part. It is related to the highly conserved DNA-binding domain of the transcription factors of the C6 class of the ascomycetes (23). At variance with other motifs of this class, the putativ...

The alcR positive control gene is necessary for the expression of both alcA (coding for alcohol dehydrogenase ADH I), and aldA (coding for aldehyde dehydrogenase, AldDH) in Aspergillus nidulans. Using a cloned alcR probe and Northern blots analysis we show that: (1) alcR itself is inducible; (2) alcR inducibility depends on the expression of the alcR gene itself; and (3) alcR is subject to carbon catabolite repression and its expression is controlled by the negatively acting creA wide specificity gene. The repression of alcR is sufficient to explain the carbon catabolite repression of ADH I and AldDH.