Structural and functional characterisation of the DNA binding domain of the Aspergillus nidulans gene regulatory protein AreA.
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 (approximately 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.
Chant
Alan
A
Biophysics Laboratories, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, St Michael's Building, White Swan Road, UK.
Provatopoulou
Xenia
X
Manfield
Iain W
IW
Kneale
G Geoff
GG
D001233:Aspergillus nidulans / Q000378:metabolism*; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D014508:Urea / Q000378:metabolism
C079728:AreA protein, Aspergillus nidulans; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D014157:Transcription Factors; D014508:Urea; D004247:DNA; D010447:Peptide Hydrolases