Identifying PhosphoCTD-Associating Proteins
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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.