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United States Department of Agriculture

Agricultural Research Service

Title: The Carboxy-Terminal 23 Amino Acid Region of Aspergillus Parasiticus Aflr Is a Transcription Activation Domain

Authors
item Chang, Perng Kuang
item Yu, Jiujiang
item Bhatnagar, Deepak
item Cleveland, Thomas

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 8, 1999
Publication Date: N/A

Interpretive Summary: Aflatoxins are produced by the "yellow green" molds, such as Aspergillus parasiticus and Aspergillus flavus. The carcinogenic properties of aflatoxins in various biological systems are well documented. Several molecular approaches have been devised to curb aflatoxin contamination of commodities. In this report, we identify a function region which is required for aflatoxin synthesis. This work represents our continued effort to understand the underlying mechanisms that govern aflatoxin production. With the knowledge on how aflatoxin biosynthesis is activated by the regulatory protein, we could develop strategies to interrupt the expression of aflatoxin pathway genes, thereby preventing preharvest contamination of crops by aflatoxins. This report will also benefit our understanding of the biosafety of food-grade A. sojae/oryzae which are closely related to aflatoxigenic A. parasiticus/flavus, but are widely used in fermentation industry.

Technical Abstract: AFLR, a DNA-binding protein of 444 amino acids, transactivates the expression of genes in the aflatoxin biosynthetic pathway gene clusters in A. parasiticus, A. flavus as well as the sterigmatocystin synthesis gene cluster in A. nidulans. We showed, by fusion of various aflR coding regions to the GAL4 DNA-binding coding region, that the AFLR carboxy-terminus contained a transcription activation domain and that the internal region between the AFLR DNA-binding and transcription activation domains was required for AFLR's function. Compared to the AFLR carboxy-terminal fusion protein (AFLRC), a mutant AFLRC retained approximately 75% of the activation activity after deletion of three acidic amino acids, Asp 365, Glu366 and Glu367, in a previously identified acidic stretch. Removal of the carboxy-terminal amino acid, Glu444, did not affect the activation activity. Substitutions of acidic Glu423, Asp439, or Asp436/Asp439 with basic amino acid(s) resulted in 10 to 15-fold lower activation activities. The Asp436His mutation abolished the activation activity. Substitutions of basic His428, and His442 with acidic Asp resulted in 20% and 40% decrease in the activation activity, respectively. Simultaneous substitutions of Arg427, Arg429, and Arg431 with Leu significantly decreased the activation activity; the decrease was approximately 50-fold. Results suggest that the carboxy-terminal 23 amino acid region of Aspergillus parasiticus AFLR is a transcription activation domain and that total acidity in this region is not a major determinant of AFLR's activation ability.

Last Modified: 4/20/2014
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