Affinity Selection of DNA-binding Protein Complexes Using mRNA Display

Nucleic Acids Res. 2006 Feb 14;34(3):e27. doi: 10.1093/nar/gnj025.

Abstract

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.

Publication types

  • Evaluation Study

MeSH terms

  • Animals
  • Binding Sites
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / isolation & purification*
  • DNA-Binding Proteins / metabolism
  • Gene Library*
  • Mice
  • Polymerase Chain Reaction
  • Protein Biosynthesis
  • Proto-Oncogene Proteins c-fos / genetics
  • Proto-Oncogene Proteins c-fos / isolation & purification
  • Proto-Oncogene Proteins c-fos / metabolism
  • Proto-Oncogene Proteins c-jun / genetics
  • Proto-Oncogene Proteins c-jun / isolation & purification
  • Proto-Oncogene Proteins c-jun / metabolism
  • RNA, Messenger / analysis*
  • Response Elements
  • Transcription Factors / genetics
  • Transcription Factors / isolation & purification*
  • Transcription Factors / metabolism

Substances

  • DNA-Binding Proteins
  • Proto-Oncogene Proteins c-fos
  • Proto-Oncogene Proteins c-jun
  • RNA, Messenger
  • Transcription Factors