Kulmburg P, Sequeval D, Lenouvel F, Mathieu M, Felenbok B
Institut de Génétique et Microbiologie, Université Paris-Sud, Orsay, France.
Mol Cell Biol. 1992 May;12(5):1932-9. doi: 10.1128/mcb.12.5.1932-1939.1992.
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.
ALCR蛋白是丝状真菌构巢曲霉乙醇利用途径的转录激活因子。这种激活因子属于一类真菌蛋白家族，其具有一个保守的DNA结合结构域，该结构域含有六个半胱氨酸（C6类），具有一些显著特征。与该类别的其他基序不同，ALCR的结合结构域相对于中心半胱氨酸具有强烈的不对称性，而且预计会采用螺旋-转角-螺旋结构。ALCR的这一结构域在大肠杆菌中合成，并作为谷胱甘肽-S-转移酶融合蛋白进行纯化。我们的结果表明，转录激活因子ALCR是一种DNA结合蛋白。DNA结合基序含有锌，这是特异性DNA结合所必需的。ALCR肽以不同亲和力与alcR编码区上游的两个特定靶点结合，这两个靶点的特征是具有保守的5核苷酸核心序列5'-CCGCA-3'（或其反向序列）。一个位点，即低亲和力结合位点，是一个直接重复序列，另一个位点，即高亲和力结合位点，是一个具有二重对称的回文序列。因此，ALCR结合蛋白能够以两种不同的构型识别一个DNA序列。通过缺失alcR基因顺式作用区域中的两个特定靶点获得的alcR突变体无法在乙醇上生长，并且不表达任何乙醇脱氢酶活性。这些结果表明，这些结合位点是构巢曲霉中ALCR蛋白在体内的功能靶点（UASalc）。它们证实了之前关于alcR是自我调节的证据。
