Published online 14 February 2006
Methods Online |
Affinity selection of DNA-binding protein complexes using mRNA display
Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
*To whom correspondence should be addressed. Tel: +81 45 566 1775; Fax: +81 45 566 1440; Email: hyana{at}bio.keio.ac.jp
Received October 31, 2005. Revised December 21, 2005. Accepted January 27, 2006.
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.
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