Journal Article

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

Papantonis, Argyris, Baboo, Sabyasachi, Deng, Binwei, Melnik, Svitlana, Carr, Ian M, Cook, Peter R

2011

Overview

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 .

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 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