Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.
Fiche publication
Date publication
février 2015
Journal
Gene
Auteurs
Membres identifiés du Cancéropôle Est :
Dr LEFEBVRE Olivier
Tous les auteurs :
Nguyen NT, Saguez C, Conesa C, Lefebvre O, Acker J
Lien Pubmed
Résumé
To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.
Mots clés
Chromatin, chemistry, Chromatography, Affinity, methods, Cross-Linking Reagents, pharmacology, Gene Regulatory Networks, Mass Spectrometry, Protein Binding, RNA Polymerase III, chemistry, Saccharomyces cerevisiae, genetics, Saccharomyces cerevisiae Proteins, chemistry, Transcription Factors, chemistry, Transcription Initiation, Genetic
Référence
Gene. 2015 Feb;556(1):51-60