Functional characterization of small CTD phosphatases.
The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase (RNAP) II undergoes reversible phosphorylation with each round of transcription essential for the regulation of gene expression. A family of small CTD phosphatases (SCPs) was identified based on a homology search to TFIIF-associating CTD phosphatase 1 (FCP1). Unlike FCP1, SCP preferentially catalyze the dephosphorylation of Ser5 within the CTD and is especially active toward RNAP II phosphorylated by TFIIH (1). Recently, SCP1 was demonstrated as a transcriptional regulator that acts to silence neuronal genes (2). This chapter describes the procedures for various assays involved in the discovery and functional characterization of SCPs.
Yeo
Michele
M
Division of Neurology, Duke University Medical Center, Durham, NC, USA.
Lin
Patrick S
PS
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D034622:RNA Interference; D012319:RNA Polymerase II / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051758:Transcription Factor TFIIH / Q000378:metabolism; D019473:Transcription Factors, TFII / Q000378:metabolism; D014158:Transcription, Genetic
D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D012319:RNA Polymerase II; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C116022:carboxy-terminal domain phosphatase; C056604:transcription factor TFIIF