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.
Phatnani
Hemali P
HP
Biochemistry Department, Duke University Medical Center, Durham, NC, USA.
Greenleaf
Arno L
AL
D015153:Blotting, Western / Q000379:methods*; D002457:Cell Extracts / Q000302:isolation & purification*; D006367:HeLa Cells; D006801:Humans; D010750:Phosphoproteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism
D002457:Cell Extracts; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; C060137:carboxy-terminal domain kinase; D012319:RNA Polymerase II