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Nat Methods. 2011 Sep 25;8(11):963-8. doi: 10.1038/nmeth.1705.

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

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1
Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.

Abstract

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.

PMID:
21946667
PMCID:
PMC3324775
DOI:
10.1038/nmeth.1705
[Indexed for MEDLINE]
Free PMC Article
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