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Identifying phosphoCTD-associating proteins.

Hemali P Phatnani·Arno L Greenleaf
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Abstract

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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Blotting, WesternCell ExtractsHeLa CellsHumansPhosphoproteinsPhosphorylationProtein BindingProtein KinasesRNA Polymerase IIRecombinant Fusion ProteinsSaccharomyces cerevisiaeJournal Article

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Hemali P Phatnani, Arno L Greenleaf. 2004. Identifying phosphoCTD-associating proteins.. https://doi.org/10.1385/1-59259-750-5%3A017

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