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

Diane Forget; Céline Domecq; Benoit Coulombe

doi:10.1007/978-1-60327-015-1_26

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

Methods Mol Biol. 2009:543:439-51. doi: 10.1007/978-1-60327-015-1_26.

Authors

Diane Forget¹, Céline Domecq, Benoit Coulombe

Affiliation

¹ Gene Transcription & Proteomics Laboratory, Institut de Recherches Cliniques de Montréal, 110, avenue des Pins ouest, Montréal, QC, Canada, H2W 1R7.

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.

MeSH terms

Animals
Autoradiography
Base Sequence
Cross-Linking Reagents / chemistry*
Cross-Linking Reagents / radiation effects*
DNA / metabolism*
Electrophoresis, Polyacrylamide Gel
Light*
Molecular Biology / methods*
Molecular Sequence Data
Proteins / metabolism
RNA Polymerase II / chemistry*
Transcription, Genetic*

Substances

Cross-Linking Reagents
Proteins
DNA
RNA Polymerase II

Grants and funding

14309-3/CAPMC/ CIHR/Canada