Use of site-specific protein-DNA photocrosslinking of purified complexes to analyze the topology of the RNA polymerase II transcription initiation complex.
A method for the photocrosslinking of proteins to DNA in purified complexes is described. It makes use of the juxtaposition of a limited number of photoreactive nucleotides with a limited number of radiolabeled nucleotides at a specific location in a DNA fragment. Protein-DNA complexes are submitted to an electrophoretic mobility shift assay that is then irradiated with UV light in order to crosslink the proteins to DNA. The specific complexes are localized on the gel, purified, and processed for the identification of the crosslinked polypeptides.
Gene Transcription & Proteomics Laboratory, Institut de Recherches Cliniques de Montréal, 110, avenue des Pins ouest, Montréal, QC, Canada, H2W 1R7.
Diane
D
Forget
Céline
C
Domecq
Benoit
B
Coulombe
D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000528:radiation effects*; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008027:Light; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism; D012319:RNA Polymerase II / Q000737:chemistry*; D014158:Transcription, Genetic
D003432:Cross-Linking Reagents; D011506:Proteins; D004247:DNA; D012319:RNA Polymerase II