The proteomes of transcription factories containing RNA polymerases I, II or III

The question of whether transcription factories containing RNA polymerases exist has been controversial, owing to the fact that they have not been isolated previously. Now, a method to carefully isolate these complexes and analyze their proteomes by mass spectrometry is described. Human nuclei conta...

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Bibliographic Details
Published in:	Nature methods Vol. 8; no. 11; pp. 963 - 968
Main Authors:	Melnik, Svitlana, Deng, Binwei, Papantonis, Argyris, Baboo, Sabyasachi, Carr, Ian M, Cook, Peter R
Format:	Journal Article
Language:	English
Published:	New York Nature Publishing Group US 01-11-2011 Nature Publishing Group
Subjects:	631/1647/2017/2214 631/1647/2067 631/1647/2230 Bioinformatics Biological Microscopy Biological Techniques Biomedical and Life Sciences Biomedical Engineering/Biotechnology Cells Deoxyribonucleic acid DNA DNA-Directed RNA Polymerases - genetics DNA-Directed RNA Polymerases - metabolism Genetic aspects HeLa Cells Humans Life Sciences Mammals Mass spectrometry Physiological aspects Proteome Proteomics RNA polymerase RNA polymerases RNA, Messenger - genetics Transcription factors Transcription, Genetic United Kingdom
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Summary:	The question of whether transcription factories containing RNA polymerases exist has been controversial, owing to the fact that they have not been isolated previously. Now, a method to carefully isolate these complexes and analyze their proteomes by mass spectrometry is described. Human nuclei contain three RNA polymerases (I, II and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the substructure. Here we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory. For example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo .
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 Present Address: Division of Molecular Biology of the Cell II, German Cancer Research Center, DKFZ-ZMBH-Alliance, INF 581, D-69120, Heidelberg, Germany AUTHOR CONTRIBUTIONS Experiments were designed by SM, BD, AP, SB, and PRC. SM developed the isolation procedure and carried out many of the validation experiments, SM and BD performed gel electrophoreses and mass spectrometry, AP developed native 3C and carried out RT-PCR, SB did the light microscopy, and IMC developed software. All authors wrote the paper.
ISSN:	1548-7091 1548-7105
DOI:	10.1038/nmeth.1705