In vitro recognition of specific DNA targets by AlcR, a zinc binuclear cluster activator different from the other proteins of this class.
AlcR is the transactivator mediating transcriptional induction of the alc gene cluster in Aspergillus nidulans. The AlcR DNA-binding domain consists of a zinc binuclear cluster different from the other members of the Zn2Cys6 family by several features. In particular, it is able to bind to symmetric and asymmetric sites with the same affinity, with both sites being functional in A. nidulans. Here, we show that unlike the other proteins of the Zn2Cys6 binuclear cluster family, AlcR binds most probably as a monomer to its cognate targets. Two molecules of the AlcR protein can simultaneously bind in a noncooperative manner to inverted repeats. The consensus core has been determined precisely (5'-CCGCN-3'), and the AlcR-binding site in the aldA promoter has been localized. The sequence downstream of the zinc cluster is necessary for high affinity binding. Furthermore, our data show that the use of the carrier protein glutathione S-transferase in AlcR binding experiments introduces an important bias in the recognition of DNA sites due to its tertiary dimeric structure.
Lenouvel
F
F
Institut de Génétique et Microbiologie, Université Paris-Sud, URA CNRS D 2225, Bâtiment 409, Centre Universitaire d'Orsay, F-91405 Orsay Cedex, France.
Nikolaev
I
I
Felenbok
B
B
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000235:genetics; D001419:Bacteria / Q000235:genetics; D004274:DNA, Recombinant / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism
C074202:ALCR protein, Aspergillus nidulans; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D015032:Zinc