Use of site-specific protein-DNA photocrosslinking of purified complexes to analyze the topology of the RNA polymerase II transcription initiation complex

Abstract

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. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.