Characterisation of the domain structure of the gene regulatory protein AreA from Aspergillus nidulans.
AreA, a 96 kDa gene regulatory protein involved in nitrogen metabolite repression in
Aspergillus nidulans, is a member of the GATA family of zinc finger DNA binding
proteins, and regulates the expression of around 100 genes. This project was designed
to examine the domain structure of AreA in this region of the protein, and to
characterise the DNA binding domain
Limited proteolysis has been employed to identify structural domains in the Cterminal
region of AreA, which has been cloned and over-produced in E.coli. A
variety of proteases have been used, and each reveals a dominant stable fragment of
approximately 17-22 kDa. N-terminal sequencing and mass spectroscopy have been
used to identify a number of these fragments.
The major product following limited proteolysis by Glu-C is composed of two closely
related species, a 164 residue fragment (17,489 Da) and a 157 residue fragment
(16,857 Da). Both fragments encompass the Zn-finger motif, and share the same Cterminus,
differing at the N-terminus by only 7 amino acids.
The DNA sequence coding for the 157 residue fragment (16,857 Da) has been cloned
and over-produced as a His-tag fusion protein. Further studies on this domain have
shown that this putative domain has a relatively strong DNA binding constant with
values in the nanomolar range. Structural analysis using Circular Dichroism, NMR
and fluorescence suggests that the domain contains some irregular or unstructured
regions. The regions that are structured are likely to be from the zinc-finger region,
since DNA binding is maintained.