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Structural and functional characterisation of the DNA binding domain of theAspergillus nidulansgene regulatory protein AreA

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Publication Date
DOI: 10.1016/s1570-9639(03)00109-2
  • Zinc Finger
  • Gata Protein
  • Fluorescence
  • Dna-Binding Domain
  • Denaturation
  • Biology


Abstract The 876-aa protein AreA regulates the expression of numerous genes involved in nitrogen metabolism in Aspergillus nidulans, and interacts with GATA sequences upstream of the relevant genes. We have carried out limited proteolysis of the C-terminal domain of the AreA protein in order to identify possible structural domains within the protein. A stable 156-amino-acid fragment was identified that contained the zinc finger region, and this sequence was cloned and expressed in E. coli. Fluorescence spectroscopy of the purified protein showed that the proteolytic domain was folded and could be denatured by high concentrations of urea (∼4 M), exhibiting a sharp transition. Fluorescence spectroscopy was also used to monitor binding to a DNA duplex containing the AreA recognition site, demonstrating tight binding of the domain to its DNA recognition sequence. The DNA binding affinity of the domain is comparable with that of the native AreA protein and much higher than that of the minimal zinc finger region of AreA.

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