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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.

Original publication

DOI

10.1038/nmeth.1705

Type

Journal article

Journal

Nat Methods

Publication Date

25/09/2011

Volume

8

Pages

963 - 968

Keywords

DNA-Directed RNA Polymerases, HeLa Cells, Humans, Proteome, RNA, Messenger, Transcription, Genetic