title: Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.
abstract: 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.
authors.affiliation: CEA, iBiTecS, SBIGeM, FRE 3377, Gif-sur-Yvette F-91191, France; CNRS, FRE 3377, Gif-sur-Yvette F-91191, France; Univ Paris-Sud, FRE 3377, Gif-sur-Yvette F-91191, France.
authors.forename: Ngoc-Thuy-Trinh
authors.initials: NT
authors.lastname: Nguyen
authors.affiliation: CEA, iBiTecS, SBIGeM, FRE 3377, Gif-sur-Yvette F-91191, France; CNRS, FRE 3377, Gif-sur-Yvette F-91191, France; Univ Paris-Sud, FRE 3377, Gif-sur-Yvette F-91191, France.
authors.forename: Cyril
authors.initials: C
authors.lastname: Saguez
authors.affiliation: CEA, iBiTecS, SBIGeM, FRE 3377, Gif-sur-Yvette F-91191, France; CNRS, FRE 3377, Gif-sur-Yvette F-91191, France; Univ Paris-Sud, FRE 3377, Gif-sur-Yvette F-91191, France.
authors.forename: Christine
authors.initials: C
authors.lastname: Conesa
authors.affiliation: CEA, iBiTecS, SBIGeM, FRE 3377, Gif-sur-Yvette F-91191, France; CNRS, FRE 3377, Gif-sur-Yvette F-91191, France; Univ Paris-Sud, FRE 3377, Gif-sur-Yvette F-91191, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Lefebvre
authors.affiliation: CEA, iBiTecS, SBIGeM, FRE 3377, Gif-sur-Yvette F-91191, France; CNRS, FRE 3377, Gif-sur-Yvette F-91191, France; Univ Paris-Sud, FRE 3377, Gif-sur-Yvette F-91191, France. Electronic address: joel.acker@cea.fr.
authors.forename: Joël
authors.initials: J
authors.lastname: Acker
mesh_terms: D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D053263:Gene Regulatory Networks; D013058:Mass Spectrometry; D011485:Protein Binding; D012320:RNA Polymerase III / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D061785:Transcription Initiation, Genetic
chemical_list: D002843:Chromatin; D003432:Cross-Linking Reagents; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D012320:RNA Polymerase III
keywords: Protein–protein interaction; RNA polymerase III; TAP-MS; TFIIIA