Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation.
Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. Numerous enzymes, including cell cycle-dependent kinases and TFIIF-dependent phosphatases target the CTD. However, the repetitive nature of the CTD prevents determination of phosphorylated sites by conventional biochemistry methods. Fortunately, a panel of monoclonal antibodies is available that distinguishes between phosphorylated isoforms of RNA polymerase II's (RNAP II) largest subunit. Here, we review how successful these tools have been in monitoring RNAP II phosphorylation changes in vivo by immunofluorescence, chromatin immunoprecipitation and immunoblotting experiments. The CTD phosphorylation pattern is precisely modified as RNAP II progresses along the genes and is involved in sequential recruitment of RNA processing factors. One of the most popular anti-phosphoCTD Igs, H5, has been proposed in several studies as a landmark of RNAP II molecules engaged in transcription. Finally, we discuss how global RNAP II phosphorylation changes are affected by the physiological context such as cell stress and embryonic development.
D000595:Amino Acid Sequence; D002843:Chromatin / Q000378:metabolism; D000939:Epitopes / Q000378:metabolism; D007527:Isoenzymes; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012319:RNA Polymerase II / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D002843:Chromatin; D000939:Epitopes; D007527:Isoenzymes; D012333:RNA, Messenger; D014157:Transcription Factors; D012694:Serine; D011494:Protein Kinases; D012319:RNA Polymerase II