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Sequence-specific binding by Aspergillus nidulans AflR, a C6 zinc cluster protein regulating mycotoxin biosynthesis
M Fernandes1, N P Keller, T H Adams
1Department of Plant Pathology and Microbiology, Texas A&M University, College Station 77843, USA.
Molecular Microbiology
|July 29, 1998
Summary
The Aspergillus nidulans aflR gene product binds to specific DNA sequences, activating genes essential for sterigmatocystin production. This binding is crucial for regulating the sterigmatocystin biosynthesis pathway in Aspergillus.
Area of Science:
- Mycology
- Molecular Biology
- Biochemistry
Background:
- The Aspergillus nidulans aflR gene is part of a 60 kb cluster containing approximately 24 genes involved in sterigmatocystin production.
- AflR is a conserved C6 zinc binuclear cluster protein acting as a pathway-specific transcription factor.
Purpose of the Study:
- To characterize the AnAflR protein and its DNA binding properties.
- To investigate the in vivo role of AnAflR binding sites in regulating sterigmatocystin (stc) gene expression.
Main Methods:
- Protein characterization of A. nidulans AflR (AnAflR).
- Electrophoretic mobility shift assays (EMSA) to identify AnAflR binding sequences.
- Gene fusion assays using the uidA (GUS) reporter gene to assess promoter activity.
- Site-directed mutagenesis of AnAflR binding elements in the stcU promoter.
Main Results:
- AnAflR is a 45kDa protein that binds to the palindromic sequence 5'-TCG(N5)CGA-3'.
- This binding site is present in the promoter regions of several aflatoxin and sterigmatocystin cluster genes.
- Activation of stc gene expression requires both AnAflR and at least one functional TCG(N5)CGA binding site.
Conclusions:
- AnAflR directly binds to specific DNA sequences to regulate sterigmatocystin biosynthesis genes.
- The presence and functionality of AnAflR binding sites are essential for stc gene activation in Aspergillus nidulans.