Identifying phosphoCTD-associating proteins.
The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is hyperphosphorylated during transcription elongation. The phosphoCTD is known to bind to a subset of RNA processing factors and to several other nuclear proteins, thereby positioning them to efficiently carry out their elongation-linked functions. The authors propose that additional phosphoCTD-associating proteins (PCAPs) exist and describe a systematic biochemical approach for identifying such proteins. A binding probe is generated by using yeast CTD kinase I to exhaustively phosphorylate a CTD fusion protein. This phosphoCTD is used to probe fractionated yeast or mammalian extracts in a Far Western protein interaction assay. Putative PCAPs are further purified and identified by mass spectrometry.
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- Saccharomyces cerevisiae
- Recombinant Fusion Proteins
- RNA Polymerase II
- Protein Kinases
- Protein Binding
- Phosphorylation
- Phosphoproteins
- Humans
- Hela Cells
- HeLa Cells
Citation
Published In
DOI
ISSN
Publication Date
Volume
Start / End Page
Location
Related Subject Headings
- Saccharomyces cerevisiae
- Recombinant Fusion Proteins
- RNA Polymerase II
- Protein Kinases
- Protein Binding
- Phosphorylation
- Phosphoproteins
- Humans
- Hela Cells
- HeLa Cells