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.
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We thank J. Bartlett for technical assistance, M. Vigneron (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg) for the 7C2 antibody, B. Thomas, D. Trudgian, G. Ridlova and M. Dreger for help with proteomics, M. Shaw for help with electron microscopy, and the Medical Research Council (S.M. and B.D.), EP Abraham Research Fund (B.D.), Biotechnology and Biological Sciences Research Council (A.P.), Wellcome Trust (A.P.) and Felix Scholarship Trust of Oxford University (S.B.) for support.
The authors declare no competing financial interests.
Supplementary Figures 1–6, Supplementary Tables 1–3, Supplementary Note (PDF 1947 kb)
Experiment 1: output from the CPFP (FDRs for complexes I, II and III were 0.84, 0.8 and 0.82% respectively), and results from the SI analysis. (XLSX 1665 kb)
Experiment 2: output from the CPFP (FDRs for complexes I, II and III were all 0.8%). (XLSX 103 kb)
Experiment 3: output from the CPFP (FDRs for complexes II and III were 0.75 and 0.65%, respectively), and results from the SI analysis. (XLSX 243 kb)
Comparison of the proteomes seen in all three experiments. (XLSX 1036 kb)
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Melnik, S., Deng, B., Papantonis, A. et al. The proteomes of transcription factories containing RNA polymerases I, II or III. Nat Methods 8, 963–968 (2011). https://doi.org/10.1038/nmeth.1705
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