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

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.

SUBMITTER: Melnik S

PROVIDER: S-EPMC3324775 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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

Melnik Svitlana S Deng Binwei B Papantonis Argyris A Baboo Sabyasachi S Carr Ian M IM Cook Peter R PR

Nature methods 20110925 11

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 conta ...[more]

PMID: 21946667

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