Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fcp1 de-phosphorylates the RNA polymerase II (RNAPII) C-terminal domain (CTD) in vitro, and mutation of the yeast FCP1 gene results in global transcription defects and increased CTD phosphorylation levels in vivo. Here we show that the Fcp1 protein associates with elongating RNAPII holoenzyme in vitro. Our data suggest that the association of Fcp1 with elongating polymerase results in CTD de-phosphorylation when the native ternary RNAPII0-DNA-RNA complex is disrupted. Surprisingly, highly purified yeast Fcp1 dephosphorylates serine 5 but not serine 2 of the RNAPII CTD repeat. Only free RNAPII0(Ser-5) and not RNAPII0-DNA-RNA ternary complexes act as a good substrate in the Fcp1 CTD de-phosphorylation reaction. In contrast, TFIIH CTD kinase has a pronounced preference for RNAPII incorporated into a ternary complex. Interestingly, the Fcp1 reaction mechanism appears to entail phosphoryl transfer from RNAPII0 directly to Fcp1. Elongator fails to affect the phosphatase activity of Fcp1 in vitro, but genetic evidence points to a functional overlap between Elongator and Fcp1 in vivo. Genetic interactions between Elongator and a number of other transcription factors are also reported. Together, these results shed new light on mechanisms that drive the transcription cycle and point to a role for Fcp1 in the recycling of RNAPII after dissociation from active genes.
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PMID:Interaction of Fcp1 phosphatase with elongating RNA polymerase II holoenzyme, enzymatic mechanism of action, and genetic interaction with elongator. 1556 57

FCP1, a phosphatase specific of the carboxyl-terminal-domain of the large subunit of the RNA polymerase II (RNAPII), stimulates transcription elongation and it is required for general transcription and cell viability. To identify novel interacting proteins of FCP1, we used a human cell line expressing an epitope flagged FCP1 and proteins, which formed complexes with FCP1, were identified by mass spectrometry. We identified four proteins: RPB2 subunit of the RNAPII, the nuclear kinase, NDR1, the methyltransferase PRMT5 and the enhancer of rudimentary homologue (ERH) proteins. Intriguingly, both the PRMT5 and ERH proteins are interacting partners of the SPT5 elongation factor. Interactions of RPB2, ERH, NDR1 and PRMT5 with FCP1 were confirmed by co-immunoprecipitation or in vitro pull-down assays. Interaction between PRMT5 and FCP1 was further confirmed by co-immunoprecipitation of endogenous proteins. We found that FCP1 is a genuine substrate of PRMT5-methylation both in vivo and in vitro, and FCP1-associated PRMT5 can methylate histones H4 in vitro.
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PMID:Identification of proteins interacting with the RNAPII FCP1 phosphatase: FCP1 forms a complex with arginine methyltransferase PRMT5 and it is a substrate for PRMT5-mediated methylation. 1567 Aug 29

Transcription and processing of mRNA precursors are coordinated events that require numerous complex interactions to ensure that they are successfully executed. We described previously an unexpected association between a transcription factor, PC4 (or Sub1 in yeast), and an mRNA polyadenylation factor, CstF-64 (Rna15 in yeast), and provided evidence that this was important for efficient transcription elongation. Here we provide insight into the mechanism by which this occurs. We show that Sub1 and Rna15 are recruited to promoters and present along the length of several yeast genes. Allele-specific genetic interactions between SUB1 and genes encoding an RNA polymerase II (RNAP II)-specific kinase (KIN28) and phosphatase (FCP1) suggest that Sub1 influences and/or is sensitive to the phosphorylation status of elongating RNAP II. Remarkably, we find that cells lacking Sub1 display decreased accumulation of Fcp1, altered RNAP II phosphorylation and decreased crosslinking of RNAP II to transcribed genes. Our data provide evidence that Rna15 and Sub1 are present along the length of several genes and that Sub1 facilitates elongation by influencing enzymes that modify RNAP II.
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PMID:The transcriptional coactivator PC4/Sub1 has multiple functions in RNA polymerase II transcription. 1569 59

FCP1, a phosphatase specific for the carboxyl-terminal domain of the largest subunit of RNA polymerase II, is regulated by the HIV-1 Tat protein, CK2, TFIIB, and the large subunit of TFIIF (RAP74). We have characterized the interactions of Tat and RAP74 with the BRCT-containing central domain of FCP1 (FCP1(562)(-)(738)). We demonstrated that FCP1 is required for Tat-mediated transactivation in vitro and that amino acids 562-685 of FCP1 are necessary for Tat interaction in yeast two-hybrid studies. From sequence alignments, we identified a conserved acidic/hydrophobic region in FCP1 adjacent to its highly conserved BRCT domain. In vitro binding studies with purified proteins indicate that HIV-1 Tat interacts with both the acidic/hydrophobic region and the BRCT domain of FCP1, whereas RAP74(436)(-)(517) interacts solely with a portion of the acidic/hydrophobic region containing a conserved LXXLL-like motif. HIV-1 Tat inhibits the binding of RAP74(436)(-)(517) to FCP1. In a companion paper (K. Abbott et al. (2005) Enhanced Binding of RNAPII CTD Phosphatase FCP1 to RAP74 Following CK2 Phosphorylation, Biochemistry 44, 2732-2745, we identified a novel CK2 site adjacent to this conserved LXXLL-like motif. Phosphorylation of FCP1(562)(-)(619) by CK2 at this site increases binding to RAP74(436)(-)(517), but this phosphorylation is inhibited by Tat. Our results provide insights into the mechanisms by which Tat inhibits the FCP1 CTD phosphatase activity and by which FCP1 mediates transcriptional activation by Tat. In addition to increasing our understanding of the role of HIV-1 Tat in transcriptional regulation, this study defines a clear role for regions adjacent to the BRCT domain in promoting important protein-protein interactions.
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PMID:Interactions of the HIV-1 Tat and RAP74 proteins with the RNA polymerase II CTD phosphatase FCP1. 1572 17

FCP1 (TFIIF-associated CTD phosphatase) is the first identified CTD-specific phosphatase required to recycle RNA polymerase II (RNAP II). FCP1 activity has been shown to be regulated by the general transcription factors TFIIF (RAP74) and TFIIB, protein kinase CK2 (CK2), and the HIV-1 transcriptional activator Tat. Phosphorylation of FCP1 by CK2 stimulates FCP1 phosphatase activity and enhances binding of RAP74 to FCP1. We have examined consensus CK2 phosphorylation sites (acidic residue n + 3 to serine or threonine residue) located immediately adjacent to both RAP74-binding sites of FCP1. We demonstrate that both of these consensus CK2 sites can be phosphorylated in vitro and that phosphorylation at either CK2 site results in enhanced binding of RAP74 to FCP1. The CK2 site adjacent to the RAP74-binding site in the central domain of FCP1 is phosphorylated at a single threonine site (T584). The CK2 site adjacent to the RAP74-binding site in the carboxyl-terminal domain can be phosphorylated at three successive serine residues (S942-S944), with phosphorylations at S942 and S944 both contributing to enhanced binding to RAP74. With the use of tandem Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR), we demonstrate that the phosphorylation of S942-S944 occurs in a semiordered fashion with the initial phosphorylation occurring at either S942 or S944 followed by a second phosphorylation to yield the S942/S944 diphosphorylated species. Using nuclear magnetic resonance (NMR) spectroscopy, we identify and map chemical shift changes onto the solution structure of the carboxyl-terminal domain of RAP74 (RAP74(436)(-)(517)) on complexation of RAP74(436)(-)(517) with phosphorylated FCP1 peptides. These results provide new functional and structural information on the role of phosphorylation in the recognition of acidic-rich activation domains involved in transcriptional regulation, and bring insights into how CK2 and TFIIF regulate FCP1 function.
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PMID:Enhanced binding of RNAP II CTD phosphatase FCP1 to RAP74 following CK2 phosphorylation. 1572 18

Reversible phosphorylation of RNA polymerase (RNAP) II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. The CTD phosphatase, FCP1, catalyzes the dephosphorylation of RNAP II and is thought to play a major role in polymerase recycling. In this study, we isolated a novel phosphatase gene by large-scale sequencing analysis of a human fetal brain cDNA library. Its cDNA is 2215 bp in length, encoding a 318-amino acid polypeptide that contains a ubiquitin-like domain and a CTD phosphatase domain. Therefore, it was termed ubiquitin-like domain containing CTD phosphatase 1 (UBLCP1). Reverse transcription PCR (RT-PCR) revealed that UBLCP1 was expressed with relatively lower levels in most adult normal tissues and higher levels in fast growing or tumor tissues. Transient transfection experiment suggested that UBLCP1 was localized in the nucleus of COS-7 cells. Significantly, UBLCP1 could dephosphorylate GST-CTD in vitro. Accordingly, UBLCP1 may play a role in the regulation of phosphorylation state of RNA polymerase II C-terminal domain.
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PMID:Cloning and characterization of a novel RNA polymerase II C-terminal domain phosphatase. 1588 30

The phosphorylation of C-terminal domain (CTD) of Rpb1p, the largest subunit of RNA polymerase II plays an important role in transcription and the coupling of various cellular events to transcription. In this study, its role in DNA damage response is closely examined in Saccharomyces cerevisiae, focusing specifically on several transcription factors that mediate or respond to the phosphorylation of the CTD. CTDK-1, the pol II CTD kinase, FCP1, the CTD phosphatase, ESS1, the CTD phosphorylation dependent cis-trans isomerase, and RSP5, the phosphorylation dependent pol II ubiquitinating enzyme, were chosen for the study. We determined that the CTD phosphorylation of CTD, which occurred predominantly at serine 2 within a heptapeptide repeat, was enhanced in response to a variety of sources of DNA damage. This modification was shown to be mediated by CTDK-1. Although mutations in ESS1 or FCP1 caused cells to become quite sensitive to DNA damage, the characteristic pattern of CTD phosphorylation remained unaltered, thereby implying that ESS1 and FCP1 play roles downstream of CTD phosphorylation in response to DNA damage. Our data suggest that the location or extent of CTD phosphorylation might be altered in response to DNA damage, and that the modified CTD, ESS1, and FCP1 all contribute to cellular survival in such conditions.
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PMID:Role of RNA polymerase II carboxy terminal domain phosphorylation in DNA damage response. 1641 Jul 68

An Arabidopsis (Arabidopsis thaliana) multigene family (predicted to be more than 20 members) encodes plant C-terminal domain (CTD) phosphatases that dephosphorylate Ser residues in tandem heptad repeat sequences of the RNA polymerase II C terminus. CTD phosphatase-like (CPL) isoforms 1 and 3 are regulators of osmotic stress and abscisic acid (ABA) signaling. Evidence presented herein indicates that CPL3 and CPL4 are homologs of a prototype CTD phosphatase, FCP1 (TFIIF-interacting CTD-phosphatase). CPL3 and CPL4 contain catalytic FCP1 homology and breast cancer 1 C terminus (BRCT) domains. Recombinant CPL3 and CPL4 interact with AtRAP74, an Arabidopsis ortholog of a FCP1-interacting TFIIF subunit. A CPL3 or CPL4 C-terminal fragment that contains the BRCT domain mediates molecular interaction with AtRAP74. Consistent with their predicted roles in transcriptional regulation, green fluorescent protein fusion proteins of CPL3, CPL4, and RAP74 all localize to the nucleus. cpl3 mutations that eliminate the BRCT or FCP1 homology domain cause ABA hyperactivation of the stress-inducible RD29a promoter, whereas RNAi suppression of CPL4 results in dwarfism and reduced seedling growth. These results indicate CPL3 and CPL4 are a paralogous pair of general transcription regulators with similar biochemical properties, but are required for the distinct developmental and environmental responses. CPL4 is necessary for normal plant growth and thus most orthologous to fungal and metazoan FCP1, whereas CPL3 is an isoform that specifically facilitates ABA signaling.
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PMID:Arabidopsis carboxyl-terminal domain phosphatase-like isoforms share common catalytic and interaction domains but have distinct in planta functions. 1690 68

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.
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PMID:Functional characterization of small CTD phosphatases. 1720 May 73

The androgen receptor (AR) is a ligand-activated transcription factor that mediates the actions of the steroid hormones testosterone and dihydrotestosterone at the level of gene transcription. The main transactivation function is modular in structure, maps to the N-terminal domain (NTD), and is termed AF1. This region of the AR is structurally flexible and functions in multiple protein-protein interactions with coregulatory proteins and components of the general transcription machinery. Using surface plasmon resonance, the binding kinetics for the interaction of AR-AF1 with the large subunit of the general transcription factor TFIIF, termed RAP74, and the coactivator SRC-1a were measured. AR-AF1 interacts with both the NTD and CTD of RAP74 and the CTD of SRC-1a. The dissociation constants ( Kd) for the binding of polypeptides derived from RAP74 are in the submicromolar range, while a peptide from SRC-1a bound with a Kd of 14 microM. Significantly, the individual NTD and CTD of RAP74 interacted with AR-AF1 with distinct binding kinetics, with the NTD exhibiting slower on and off rates. TFIIF is involved in transcription initiation and elongation, and the CTD of RAP74 binds to the RNA polymerase II enzyme, the general transcription factor TFIIB, and a CTD phosphatase, FCP1. We have mutated hydrophobic residues in the RAP74-CTD structure to disrupt secondary structure elements and show that binding of AR-AF1 depends upon helix 3 in the winged-helix domain of the RAP74-CTD polypeptide. Altogether, a model is suggested for AR-AF1-dependent transactivation of receptor-target genes.
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PMID:Functional characterization of the native NH2-terminal transactivation domain of the human androgen receptor: binding kinetics for interactions with TFIIF and SRC-1a. 1828 9


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