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Updated: Nov 23, 2025

High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
Published on: February 7, 2019
Biophysical characterization of the complex between the iron-responsive transcription factor Fep1 and DNA
Adriana E Miele1,2, Laura Cervoni1, Aline Le Roy3
1Department of Biochemical Sciences 'A. Rossi Fanelli', Sapienza University of Rome, P.le Aldo Moro 5, 00185, Rome, Italy.
Abstract:
Fep1 is an iron-responsive GATA-type transcriptional repressor present in numerous fungi. The DNA-binding domain of this protein is characterized by the presence of two zinc fingers of the Cys2-Cys2 type and a Cys-X5-Cys-X8-Cys-X2-Cys motif located between the two zinc fingers, that is involved in binding of a [2Fe-2S] cluster. In this work, biophysical characterization of the DNA-binding domain of Pichia pastoris Fep1 and of the complex of the protein with cognate DNA has been undertaken. The results obtained by analytical ultracentrifugation sedimentation velocity, small-angle X-ray scattering and differential scanning calorimetry indicate that Fep1 is a natively unstructured protein that is able to bind DNA forming 1:1 and 2:1 complexes more compact than the individual partners. Complex formation takes place independently of the presence of a stoichiometric [2Fe-2S] cluster, suggesting that the cluster may play a role in recruiting other protein(s) required for regulation of transcription in response to changes in intracellular iron levels.
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