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Mol Cell. 2016 Jan 21;61(2):297-304. doi: 10.1016/j.molcel.2015.12.021.

Direct Analysis of Phosphorylation Sites on the Rpb1 C-Terminal Domain of RNA Polymerase II.

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1
Department of Biochemical Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Department of Cancer Biology and Blais Proteomics Center, Dana Farber Cancer Institute, Boston, MA 02115, USA.
2
Department of Biochemical Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
3
Department of Biochemical Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: steveb@hms.harvard.edu.

Abstract

Dynamic interactions between RNA polymerase II and various mRNA-processing and chromatin-modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code."

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PMID:
26799764
PMCID:
PMC4724063
DOI:
10.1016/j.molcel.2015.12.021
[Indexed for MEDLINE]
Free PMC Article
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