Biochemical and structural studies of the RNAPII CTD phosphatase FCP1
Abbott, Karen Lynn
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FCP1 (TFIIF-associated C-terminal domain phosphatase) is the first identified phosphatase specific for the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII). FCP1 is an essential component of the RNAPII holoenzyme that plays important roles in transcription elongation as well as polymerase recycling. Human FCP1 can be divided into three main domains based on sequence homology with yeast: the amino-terminal FCP1 homology domain (FCPH), the central domain containing a single BRCT (BRCA1 C-terminus) repeat, and the highly acidic carboxyl-terminal domain. Regulation of FCP1 phosphatase activity is complex involving multiple protein-protein interactions. The large subunit of TFIIF (RAP74) interacts directly with two conserved hydrophobic motifs located in the central and carboxyl-terminal domains of FCP1 to stimulate the phosphatase activity which is vital for polymerase recycling. Stimulation by RAP74 can be inhibited by the general transcription factor TFIIB. The HIV-1 transcriptional activator Tat has also been shown to interact with the central domain of FCP1, and this interaction inhibits phosphatase activity in vitro. FCP1 is a phosphoprotein, and phosphorylation by protein casein kinase II (CK2) stimulates phosphatase activity in vitro. We have used biochemical and NMR-based structural studies to analyze the interactions of the FCP1 central domain with HIV-1 Tat, RAP74, CK2, and nucleic acids. This work has identified and characterized a conserved region rich in acidic and hydrophobic amino acids adjacent to the BRCT repeat within the central domain of FCP1 important for interactions with RAP74 and HIV-1 Tat. Tat binds FCP1 at two distinct non-overlapping sites located within the acidic/hydrophobic region and the BRCT domain. A novel threonine CK2 phosphorylation site is identified between these two Tat-binding sites, and binding of HIV-1 Tat inhibits phosphorylation by CK2. Nucleic acid-binding activity was identified for the central domain of FCP1, and this led to the discovery of a novel endonuclease activity. This work has illuminated novel activities for FCP1 and suggests that FCP1 may have additional roles in repair, termination, and/or mRNA processing events.