Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans.
The ALCR protein is the transcriptional activator of the ethanol utilization pathway in the filamentous fungus Aspergillus 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 (UASalc) for the ALCR protein in A. nidulans. They corroborate prior evidence that alcR is autoregulated.
Kulmburg
P
P
Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France.
Sequeval
D
D
Lenouvel
F
F
Mathieu
M
M
Felenbok
B
B
D001233:Aspergillus nidulans / Q000235:genetics*; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D015342:DNA Probes; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D016335:Zinc Fingers / Q000235:genetics
C074202:ALCR protein, Aspergillus nidulans; D015342:DNA Probes; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase