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Methods Mol Biol. 2004;257:17-28.

Identifying phosphoCTD-associating proteins.

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1
Biochemistry Department, Duke University Medical Center, Durham, NC, USA.

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.

PMID:
14769993
DOI:
10.1385/1-59259-750-5:017
[Indexed for MEDLINE]
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