Affinity selection of DNA-binding protein complexes using mRNA display.
Comprehensive analysis of DNA-protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro selection of DNA-binding protein heterodimeric complexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultaneously enriched about 100-fold from a model library (a 1:1:20 000 mixture of c-fos, c-jun and gst genes) after one round of selection. Furthermore, almost all kinds of the AP-1 family genes including c-jun, c-fos, junD, junB, atf2 and b-atf were successfully selected from an mRNA display library constructed from a mouse brain poly A(+) RNA after six rounds of selection. These results indicate that the mRNA display selection system can identify a variety of DNA-binding protein complexes in a single experiment. Since almost all transcription factors form heterooligomeric complexes to bind with their target DNA, this method should be most useful to search for DNA-binding transcription factor complexes.
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
Seiji
S
Tateyama
Kenichi
K
Horisawa
Hideaki
H
Takashima
Etsuko
E
Miyamoto-Sato
Nobuhide
N
Doi
Hiroshi
H
Yanagawa
D000818:Animals; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015723:Gene Library; D051379:Mice; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*; D020218:Response Elements; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D004268:DNA-Binding Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D014157:Transcription Factors