Comprehensive analysis of the DNA-binding specificity of an Aspergillus nidulans transcription factor, AmyR, using a bead display system.
The in vitro DNA binding profile of Aspergillus nidulans transcription factor AmyR was analyzed by a novel approach employing a genetic library of beads and flow cytometry analysis. An artificial library with 22 randomized nucleotides was constructed and subjected to a protein-DNA binding reaction with MalE-tagged AmyR. DNA fragments with potential AmyR-binding sites were labeled with fluorescence-conjugated antibody to be enriched by flow cytometry through 5 rounds of successive selection. Finally, a binding motif with a single CGG triplet was obtained from DNA fragments showing weak AmyR binding, while another motif with dual CGG triplets was discovered with stronger binding fragments. An informative motif, CGGNNNTTTNTCGG, was found to exist only in the promoter region of highly AmyR-dependent genes. These results suggest that this system is a powerful tool for the rapid and comprehensive analysis of the binding preferences of transcription factors.
Department of Bioengineering Sciences, Graduate School of Bioagricultural Sciences, Nagoya University.
Panhui
P
Wang
Takaaki
T
Kojima
Tetsuo
T
Kobayashi
Hideo
H
Nakano
D000906:Antibodies / Q000737:chemistry; D001233:Aspergillus nidulans / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D052156:SELEX Aptamer Technique; D013194:Staining and Labeling; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D000906:Antibodies; D004271:DNA, Fungal; D005656:Fungal Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D015534:Trans-Activators; C414005:amyR protein, Aspergillus