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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:369475 |
| Date | January 2001 |
| Creators | Chant, Alan |
| Publisher | University of Portsmouth |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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