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• W1996658265 abstract "TFIIF (RAP30/74) is a general initiation factor that also increases the rate of elongation by RNA polymerase II. A two-hybrid screen for RAP74-interacting proteins produced cDNAs encoding FCP1a, a novel, ubiquitously expressed human protein that interacts with the carboxyl-terminal evolutionarily conserved domain of RAP74. Related cDNAs encoding FCP1b lack a carboxyl-terminal RAP74-binding domain of FCP1a. FCP1 is an essential subunit of a RAP74-stimulated phosphatase that processively dephosphorylates the carboxyl-terminal domain of the largest RNA polymerase II subunit. FCP1 is also a stoichiometric component of a human RNA polymerase II holoenzyme complex. TFIIF (RAP30/74) is a general initiation factor that also increases the rate of elongation by RNA polymerase II. A two-hybrid screen for RAP74-interacting proteins produced cDNAs encoding FCP1a, a novel, ubiquitously expressed human protein that interacts with the carboxyl-terminal evolutionarily conserved domain of RAP74. Related cDNAs encoding FCP1b lack a carboxyl-terminal RAP74-binding domain of FCP1a. FCP1 is an essential subunit of a RAP74-stimulated phosphatase that processively dephosphorylates the carboxyl-terminal domain of the largest RNA polymerase II subunit. FCP1 is also a stoichiometric component of a human RNA polymerase II holoenzyme complex. RNA polymerase general transcription factor for RNA polymerase II carboxyl-terminal domain of the largest subunit of RNA polymerase II unphosphorylated form of RNA polymerase II hyperphosphorylated form of RNA polymerase II positive transcription elongation factor human immunodeficiency virus, type 1 transactivator protein of HIV-1 RNA polymerase II associating protein TFIIF-associating CTD phosphatase rapid amplification of mRNA ends glutathioneS-transferase 3-aminotriazole polymerase chain reaction affinity chromatography buffer 3-[(3-cholamidopropyl)dimethylammonio]-1-pro-panesulfonic acid DNA-binding domain. Initiation of transcription by RNA polymerase (RNAP)1 II involves the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (reviewed in Ref. 1Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (844) Google Scholar). Beginning with TFIID, whose TATA box-binding protein subunit recognizes the TATA box present in many promoters, these factors can assemble in an ordered pathway in vitro onto a promoter (2Van Dyke M.W. Roeder R.G. Sawadogo M. Science. 1988; 241: 1335-1338Crossref PubMed Scopus (194) Google Scholar, 3Buratowski S. Hahn S. Guarente L. Sharp P.A. Cell. 1989; 56: 549-561Abstract Full Text PDF PubMed Scopus (680) Google Scholar), resulting in the formation of a preinitiation complex containing more than 40 polypeptides. Subsequently, however, yeast and mammalian RNAP II holoenzymes that contain several or all of the general transcription factors and other polypeptides were discovered (4Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Crossref PubMed Scopus (530) Google Scholar, 5Kim Y.-J. Björklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (884) Google Scholar, 6Ossipow V. Tassan J.-P. Nigg E.A. Schibler U. Cell. 1995; 83: 137-146Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 7Chao D. Gadbois E.L. Murray P.J. Anderson S.F. Sonu M.S. Parvin J.D. Young R.A. Nature. 1996; 380: 82-85Crossref PubMed Scopus (127) Google Scholar, 8Maldonado E. Shiekhattar R. Sheldon M. Cho H. Drapkin R. Rickert P. Lees E. Anderson C.W. Linn S. Reinberg D. Nature. 1996; 381: 86-89Crossref PubMed Scopus (306) Google Scholar, 9Pan G. Aso T. Greenblatt J. J. Biol. Chem. 1997; 272: 24563-24571Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). There is evidence that transcription by RNAP II in Saccharomyces cerevisiaegenerally depends on such a holoenzyme (10Thompson C.M. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4587-4590Crossref PubMed Scopus (208) Google Scholar) and that recruitment of yeast holoenzyme to a promoter would lead to a high rate of transcription (11Barberis A. Pearlberg J. Simkovich N. Farrell S. Reinagel P. Bamdad C. Sigal G. Ptashne M. Cell. 1995; 81: 359-368Abstract Full Text PDF PubMed Scopus (235) Google Scholar). During or shortly after initiation by RNAP II, the carboxyl-terminal domain (CTD) of its largest subunit becomes heavily phosphorylated and remains so during transcript elongation (12Payne J.M. Laybourn P.J. Dahmus M.E. J. Biol. Chem. 1989; 264: 19621-19629Abstract Full Text PDF PubMed Google Scholar). The phosphorylated form of RNAP II is designated RNAP IIO, whereas the unphosphorylated form is designated RNAP IIA. One subunit of TFIIH is a protein kinase that can phosphorylate the CTD (13Emili A. Ingles C.J. Curr. Opin. Genet. Dev. 1995; 5: 204-209Crossref PubMed Scopus (19) Google Scholar). Phosphorylation of the CTD by P-TEFb, a different Drosophila CTD kinase, has been shown to enhance the processivity of chain elongation by RNAP II in vitro(14Marshall N.F. Peng J. Xie Z. Price D.H. J. Biol. Chem. 1996; 271: 27176-27183Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). Concomitant with or following the termination of transcription, the CTD must be dephosphorylated by a protein phosphatase, since RNAP IIO cannot assemble directly into a preinitiation complex on either the adenovirus-2 major late or murine dihydrofolate reductase promoterin vitro (15Lu H. Flores O. Weinmann R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10004-10008Crossref PubMed Scopus (248) Google Scholar, 16Chesnut J.D. Stephens J.H. Dahmus M.E. J. Biol. Chem. 1992; 267: 10500-10506Abstract Full Text PDF PubMed Google Scholar, 17Kang M.E. Dahmus M.E. J. Biol. Chem. 1993; 268: 25033-25040Abstract Full Text PDF PubMed Google Scholar). Accordingly, CTD phosphatase may function as a global regulator of gene expression by controlling the pool of RNAP IIA available for initiation. A phosphatase whose activity is stimulated by RAP74 and dephosphorylates the CTD in a processive manner has been purified from HeLa cell extracts (18Chambers R.S. Dahmus M.E. J. Biol. Chem. 1994; 269: 26243-26248Abstract Full Text PDF PubMed Google Scholar, 19Chambers R.S. Wang B.Q. Burton Z.F. Dahmus M.E. J. Biol. Chem. 1995; 270: 14962-14969Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Certain activator proteins increase the efficiency of RNA chain elongation downstream from the promoter. For example, RNAP II pauses with an unphosphorylated CTD about 25–40 nucleotides downstream from the initiation site of Drosophila hsp70 genes and is stimulated by heat shock and the heat shock factor to become phosphorylated and leave these pause sites (Ref. 20O'Brien T. Hardin S. Greenleaf A. Lis J.T. Nature. 1994; 370: 75-77Crossref PubMed Scopus (285) Google Scholar and references therein). Increasing evidence supports the idea that a fully phosphorylated CTD is essential for processive elongation. Indeed, the ability of an activator to stimulate elongation has correlated with its ability to bind TFIIH (21Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles C.J. Greenblatt J. Mol. Cell. Biol. 1994; 14: 7013-7024Crossref PubMed Scopus (327) Google Scholar, 22Blau J. Xiao H. McCracken S. O'Hare P. Greenblatt J. Bentley D. Mol. Cell. Biol. 1996; 16: 2044-2055Crossref PubMed Scopus (235) Google Scholar). As well, the CTD kinase, P-TEFb, is needed for activation of elongation by the HIV-1 transactivator Tat (23Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (609) Google Scholar), and Tat can stimulate phosphorylation of the CTD by TFIIHin vitro (24Parada C.A. Roeder R.G. Nature. 1996; 384: 375-378Crossref PubMed Scopus (237) Google Scholar). In concert with CTD kinases that act on RNAP II in an elongation complex, CTD phosphatase may also play a role in the regulation of transcript elongation. TFIIF is a general transcription factor comprised of two subunits, RAP30 and RAP74, and both subunits mediate its interaction with RNAP II (25Sopta M. Carthew R.W. Greenblatt J. J. Biol. Chem. 1985; 260: 10353-10360Abstract Full Text PDF PubMed Google Scholar, 26Kephart D.D. Wang B.Q. Burton Z.F. Price D.H. J. Biol. Chem. 1994; 269: 13536-13543Abstract Full Text PDF PubMed Google Scholar, 27Kang M.E. Dahmus M.E. J. Biol. Chem. 1995; 270: 23390-23397Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 28Killeen M. Greenblatt J. Mol. Cell. Biol. 1992; 12: 30-37Crossref PubMed Scopus (57) Google Scholar, 29McCracken S. Greenblatt J. Science. 1991; 253: 900-902Crossref PubMed Scopus (72) Google Scholar, 30Fang S.M. Burton Z.F. J. Biol. Chem. 1996; 271: 11703-11709Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). TFIIF regulates elongation as well as initiation by RNAP II (31Burton Z.F. Killeen M. Sopta M. Ortolan L.G. Greenblatt J. Mol. Cell. Biol. 1988; 8: 1602-1613Crossref PubMed Scopus (79) Google Scholar, 32Price D.H. Sluder A.E. Greenleaf A.L. Mol. Cell. Biol. 1989; 9: 1465-1475Crossref PubMed Scopus (133) Google Scholar, 33Bengal E. Flores O. Krauskopf A. Reinberg D. Aloni Y. Mol. Cell. Biol. 1991; 11: 1195-1206Crossref PubMed Scopus (116) Google Scholar). In an attempt to identify novel factors that regulate elongation by RNAP II, a two-hybrid screen in S. cerevisiae(34Fields S. Song O.K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4838) Google Scholar) was used to clone human proteins that interact with RAP74. This article describes the molecular cloning and properties of FCP1, an essential subunit of a TFIIF-associating CTDphosphatase. FCP1 also appears to be a component of a human RNAP II holoenzyme complex. Yeast manipulations and growth media were as described previously (35Sherman F. Fink G.R. Hicks J.B. Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). To test the ability of yeast cells to grow on medium containing 3-aminotriazole (AT), 3 μl of a cell suspension (approximately 2000 cells) were applied onto SD medium containing 100 μg/ml adenine and 30 mm AT. As a control, a similar number of cells was also applied onto medium lacking AT but containing 100 μg/ml adenine and histidine. Plasmids expressing either lamin, p53, or SNF1 fused to the GAL4 AD have been described previously (34Fields S. Song O.K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4838) Google Scholar, 36Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar). pAD-FCP1a plasmids encoding amino acids 443–842, 579–842, 627–842, and 727–842 were isolated in the original two-hybrid screen. pET23d/RAP74 expressing RAP74 under the control of the T7 promoter was a gift of Z. Burton (Michigan State University) and has been described (37Wang B.Q. Kostrub C.F. Finkelstein A. Burton Z.F. Protein Expression Purif. 1993; 4: 207-214Crossref PubMed Scopus (44) Google Scholar). All other plasmids were constructed specifically for this work, and details will be provided upon request. Yeast strain Y153 (36Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar) was cotransformed to tryptophan and leucine prototrophy by introduction of plasmid pAS-RAP74 and of a human lymphocyte cDNA library constructed in plasmid pSE1107 (36Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar). Yeast were transformed as described (38Scheistl R.H. Gietz R.D. Curr. Genet. 1989; 16: 339-346Crossref PubMed Scopus (1771) Google Scholar). Transformants were selected by plating directly on SD minimal medium supplemented with adenine and 30 mm AT and grown for 7–9 days at 30 °C. Under these conditions, 50 of approximately three million transformants were able to form colonies. Library-derived plasmids were isolated from these 50 transformants, recovered in Escherichia coli, and reintroduced individually into Y153 along with pAS-RAP74 or with other plasmids encoding unrelated GAL4 DBD fusion proteins (lamin, p53, SNF1, p62 subunit of TFIIH; see Fig. 1). Of these 50 plasmids, 13 were able to support yeast growth on medium containing AT in the presence of pAS-RAP74 but not when pAS-RAP74 was substituted for another plasmid encoding an unrelated GAL4 DBD fusion protein. These 13 plasmids that encoded putative TFIIF (RAP74)-interacting proteins were grouped into the following three classes: RAP30, FCP1a, and one other on the basis of the nucleotide sequence of the encoded cDNAs. Isolation of the 5′-end of the FCP1 mRNA was performed using a 5′-rapid amplification of mRNA ends (5′-RACE; Ref. 39Frohman M. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4329) Google Scholar) protocol and a c-RACE protocol (40Maruyama I.N. Rakow T.L. Maruyama H.I. Nucleic Acids Res. 1995; 23: 3796-3797Crossref PubMed Scopus (120) Google Scholar). 5′-RACE was performed using a Marathon cDNA amplification kit (CLONTECH) following procedures recommended by the manufacturer. Briefly, oligo(dT)-primed cDNA was synthesized using poly(A)+ RNA isolated from human placenta. The first PCR amplification was performed using oligonucleotides AP1 (provided with the kit) and either FCP1–51 (5′-GCTCAGCACCAGCCGAGTGAGGATCTTCGC-3′) or FCP1–504 (5′-GCTGCCGAAGTGAAGACGTGC-3′). The second PCR amplification was performed with oligonucleotides AP2 (provided with the kit) and FCP1–52 (5′-CCGTGGCGTGGTAATCGTCCCGCGTCTTC-3′) if FCP1–51 was used for the first amplification. If FCP1–504 was used for the first amplification, the second amplification was performed using oligonucleotide AP2 and either FCP1–501 (5′-TCAGCTTGCTCGGAGCTCACC-3′) or FCP1–503 (5′-AGGAAGTCCTTGCAGTGTGGACG-3′). The 5′-end of the FCP1 mRNA was also isolated using a c-RACE protocol essentially as described (40Maruyama I.N. Rakow T.L. Maruyama H.I. Nucleic Acids Res. 1995; 23: 3796-3797Crossref PubMed Scopus (120) Google Scholar). Briefly, FCP1 cDNA was synthesized with oligonucleotide FCP1–501 and human placental poly(A)+ RNA using either Moloney murine leukemia virus (Life Technologies, Inc.) or SuperScript II (Life Technologies, Inc.) reverse transcriptase. cDNA synthesis with SuperScript II reverse transcriptase was carried out using either 0.5 or 3 mm dNTPs in the reaction. FCP1 cDNA was also synthesized from total CaCO2 RNA using avian myeloblastosis virus-reverse transcriptase (Amersham Pharmacia Biotech). All four cDNAs were subjected to a first PCR amplification using two FCP1-specific oligonucleotide primers: FCP1-C1 (5′-TGCTTCCCGTTCTTACTCTGC-3′) and FCP1-C2 (5′-CGCTGTCCACGGCGACCG-3′). The secondary PCR amplification was performed using two other FCP1-specific oligonucleotides: FCP1-C3 (5′-AACTGGGTGAGGTCTTGGCC-3′) and FCP1-C4 (5′-ATGGTGCACAGCGTCGCGG-3′. In total, three different 5-RACE conditions and four different c-RACE conditions were used. For each condition, the final PCR product was cloned into the pCR II plasmid (Invitrogen) and sequenced. All seven conditions identified the same 5′-end of the FCP1 mRNA (within 30 base pairs). The complete nucleotide sequence of the 5′-end and the remaining portion of the FCP1 cDNA was determined using a combination of deletions, commercially available primers, and primers designed on the basis of the FCP1 sequence and can be found under GenBankTM accession numberAF081287. A human multiple tissue Northern blot containing approximately 2 μg of poly(A)+ RNA isolated from the indicated tissues (CLONTECH) was probed with a FCP1a probe or a β-actin control probe (supplied by the manufacturer) as recommended by the manufacturer. The FCP1a probe was aBglII fragment from pAD-FCP1a (579–842) encoding amino acids 579–842 of FCP1a and all 3′-untranslated sequences. A plasmid pJA518 that was used to express the carboxyl terminus of FCP1a as a polyhistidine fusion in bacteria was constructed by inserting a 374-base pair StuI-AluI FCP1 fragment into the BamHI site (made blunt) of pRSET-C (Invitrogen). Plasmid pJA518 was introduced into bacterial strain JA328, which was constructed by inserting plasmid pREP4 (Qiagen), which carries the lacI gene of E. coli, into bacterial strain BL21(DE3). Overproduction of lac repressor from pREP4 further reduces the expression of T7 RNAP in BL21(DE3) cells grown under non-inducing conditions. JA328 cells carrying plasmid pJA518 were grown in Luria broth medium at 30 °C to an optical density (600 nm) of 0.5, at which point FCP1a protein synthesis was induced by the addition of 2 mm isopropyl-1-thio-b-d-galactopyranoside to the growth medium, and the cells were grown for an additional 3 h. Cells were harvested by centrifugation, and the FCP1a polyhistidine fusion protein was purified under denaturing conditions by nickel-chelate affinity chromatography on His-Bind resin (Novagen) according to the instructions supplied by the manufacturer. The purified protein was dialyzed against phosphate-buffered saline and remained completely soluble after dialysis. 40-μl columns containing GST, GST-FCP1a (amino acids 760–842), or GST-RAP74-(436–517) immobilized on glutathione-Sepharose beads at a concentration of 3–4 mg/ml (or as otherwise indicated) were prepared as described previously (41Lin Y.-S. Green M.R. Cell. 1991; 64: 971-981Abstract Full Text PDF PubMed Scopus (366) Google Scholar). Columns were first equilibrated with 200 μl of ACB buffer (10 mm HEPES, pH 7.9, 1 mm EDTA, 1 mmdithiothreitol, 10% glycerol) containing 0.1 m NaCl and then loaded with either 800 μl of HeLa whole cell extract (at a concentration of 6–8 mg/ml and prepared as described previously (25Sopta M. Carthew R.W. Greenblatt J. J. Biol. Chem. 1985; 260: 10353-10360Abstract Full Text PDF PubMed Google Scholar)) that was dialyzed against ACB buffer containing 0.1 m NaCl or with 4 μg of purified recombinant protein mixed with molecular weight markers in a total volume of 120 μl of ACB buffer containing 0.1 m NaCl. After loading, the columns were washed with 400 μl of ACB buffer containing 0.1 m NaCl. The bound proteins were eluted sequentially with 120 μl of ACB buffer containing 1 m NaCl and 120 μl of ACB buffer containing 0.1% SDS. Aliquots of the input proteins and of the NaCl and SDS eluates were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting or silver staining. Human RNAP II holoenzyme was purified from HeLa whole cell extract by affinity chromatography on GST-TFIIS and GST-elongin A-(1–123) columns and by Sepharose CL-2B chromatography as described elsewhere (9Pan G. Aso T. Greenblatt J. J. Biol. Chem. 1997; 272: 24563-24571Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Western blotting was by the enhanced chemiluminescence procedure (Amersham Pharmacia Biotech) with antibodies that have been described elsewhere (9Pan G. Aso T. Greenblatt J. J. Biol. Chem. 1997; 272: 24563-24571Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). A portion of the FCP1 open reading frame (amino acids 443–669) was expressed in bacteria as a polyhistidine fusion protein. Plasmid pJA533 that was used to express FCP1 was constructed by inserting a BglII-SmaI fragment from pAD-FCP1a-(443–842) between the BamHI and PvuII sites of pRSET-C (Invitrogen). pJA533 was introduced into bacterial strain JA328 (BL21[DE3] + pREP4). JA328 cells carrying plasmid pJA533 were grown in Luria broth medium at 30 °C to an optical density (600 nm) of 0.5 at which point FCP1 protein synthesis was induced by the addition of 2 mmisopropyl-1-thio-b-d-galactopyranoside to the growth medium, and the cells were grown for a further 3 h. Cells were harvested by centrifugation, and the FCP1 polyhistidine fusion protein was purified under denaturing conditions by nickel-chelate affinity chromatography on His-Bind resin (Novagen) according to the instructions supplied by the manufacturer. The purified protein was dialyzed against phosphate-buffered saline. Two rabbits were each immunized with 1 mg of protein emulsified in complete Freund adjuvant and boosted every 4 weeks with 0.5 mg of protein in incomplete Freund's adjuvant until a high serum titer was obtained. Anti-FCP1 antibodies were affinity purified using the antigen as described (42Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 313-315Google Scholar). 500 μl of HeLa cell nuclear transcription extract prepared as described (43Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9150) Google Scholar) were incubated with 1 μg of affinity purified anti-FCP1 antibodies or control IgG (Bio-Rad) for 1 h at 4 °C and then loaded onto 20 μl of protein A-Sepharose columns that had been prewashed with 400 μl of BC100 buffer (Ref. 44Ge H. Roeder R.G. Cell. 1994; 78: 513-523Abstract Full Text PDF PubMed Scopus (307) Google Scholar; 20 mm Tris-HCl, 0.2 mm EDTA, 0.2 mmdithiothreitol, 20% glycerol, and 100 mm KCl) containing 250 μg/ml bovine serum albumin and equilibrated with BC100 buffer before loading. The flow through fractions were aliquoted and stored at −70 °C. 32P-Labeled RNAP IIO used as substrate for CTD phosphatase assays was prepared as described by Chesnutet al. (16Chesnut J.D. Stephens J.H. Dahmus M.E. J. Biol. Chem. 1992; 267: 10500-10506Abstract Full Text PDF PubMed Google Scholar). RNAP IIO was purified by the method of Kim and Dahmus (45Kim W.-Y. Dahmus M.E. J. Biol. Chem. 1988; 263: 18880-18885Abstract Full Text PDF PubMed Google Scholar). CTD phosphatase assays were performed as described previously (18Chambers R.S. Dahmus M.E. J. Biol. Chem. 1994; 269: 26243-26248Abstract Full Text PDF PubMed Google Scholar). CTD phosphatase was purified from HeLa cells as described by Chambers and Dahmus (18Chambers R.S. Dahmus M.E. J. Biol. Chem. 1994; 269: 26243-26248Abstract Full Text PDF PubMed Google Scholar) with the following modifications. Following chromatography on HiTrap Q, which was used in place of Resource Q, fractions containing CTD phosphatase activity were pooled and loaded onto a 1.5-ml ceramic hydroxylapatite column (Bio-Rad). The column was developed with a 15-ml linear gradient of KH2PO4 from 0.012 to 0.6 m. Peak fractions from phenyl-Superose were chromatographed directly on Mono Q. The final step in the purification process was the sedimentation of CTD phosphatase on a glycerol gradient. FCP1 was also purified by a different method involving affinity chromatography on a GST-RAP74-(436–517) column. HeLa whole cell extract (100 ml) (25Sopta M. Carthew R.W. Greenblatt J. J. Biol. Chem. 1985; 260: 10353-10360Abstract Full Text PDF PubMed Google Scholar) in 50 mm Tris-Cl, pH 7.9, 100 mm KCl, 5 mm MgCl2, 0.5 mm dithiothreitol, 20% glycerol was chromatographed on a 90-ml heparin-Sepharose column and eluted with a 400-ml linear gradient to 0.5 m KCl. Pooled fractions containing FCP1 (60 ml) were brought to 0.7 m(NH4)2SO4 and loaded onto a 5-ml phenyl-Sepharose column. This column was washed first with a gradient from 0.7 m to no (NH4)2SO4 in 50 mmTris-Cl, pH 7.9, 0.5 mm dithiothreitol, 0.1 mmEDTA, 10% glycerol and then with the same gradient in buffer containing 0.05% CHAPS. The fractions containing FCP1 eluted from the phenyl-Sepharose column at the end of the second gradient and were pooled (9.8 ml) and concentrated with a Centricon concentrator to 0.9 ml. This fraction was passed successively through two 100-μl columns containing first 1 mg/ml GST and then 1 mg/ml GST-RAP74-(436–517). The columns were washed with 1 ml of affinity chromatography buffer (ACB) containing 0.1 m NaCl and 0.05% CHAPS and eluted with 50-μl aliquots of ACB containing 0.05% CHAPS and 0.5 mNaCl. Fractions containing FCP1 were pooled (100 μl). The yeast two-hybrid system (34Fields S. Song O.K. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4838) Google Scholar, 36Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1297) Google Scholar) was used to identify cDNAs in a human peripheral lymphocyte library that encodes proteins that interact with RAP74. Of approximately 3 million yeast transformants, 13 cDNAs were identified that could interact specifically with a fusion protein containing the entire RAP74 open reading frame fused to the GAL4 DNA-binding domain (DBD; amino acids 1–147) and not with several other unrelated GAL4 DBD fusion proteins (Fig. 1 a). In this assay, the protein-protein interaction was detected by the activation of aHIS3 reporter gene under the control of upstream activating sequenceGal. Increased expression of HIS3 renders yeast cells resistant to the histidine analog 3-aminotriazole (AT). Among the 13 cDNAs encoding RAP74-interacting proteins, one class comprised cDNAs encoding RAP30 (eight isolates), the small subunit of TFIIF, and one isolate was a novel, uncharacterized cDNA. The remaining four isolates contained various portions of a novel cDNA termed FCP1a (FCP: TFIIF-associating CTDphosphatase). Seven additional FCP1a cDNA clones were obtained by screening two different cDNA libraries from CaCO2 cells (a colon carcinoma cell line) and fetal brain tissue. Oligonucleotide primers were then designed and used to isolate the 5′-end of the FCP1a cDNA using either a 5′-rapid amplification of mRNA ends (5′-RACE) (39Frohman M. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4329) Google Scholar) or a c-RACE protocol (40Maruyama I.N. Rakow T.L. Maruyama H.I. Nucleic Acids Res. 1995; 23: 3796-3797Crossref PubMed Scopus (120) Google Scholar) (see “Experimental Procedures”). Both protocols identified the same 5′-end of this FCP1a cDNA. The deduced amino acid sequence of FCP1a (842 amino acids) is presented in Fig. 1 c. Northern blot analysis under high stringency conditions (Fig. 1 b, upper panel) revealed an mRNA of approximately 3.6 kilobase pairs in all human tissues that were examined (heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas). When comparison was made to the amounts of β-actin mRNA in these samples (Fig. 1 b, lower panel), results indicated that the amounts of FCP1 mRNA are of the same order of magnitude in all tissues. Other less abundant mRNAs were also visible, and under low stringency hybridization conditions, additional mRNAs of various sizes also hybridized to the FCP1 probe, suggesting that FCP1 may belong to a family of proteins (data not shown). Several FCP1 cDNAs cloned from fetal brain and CaCO2 libraries contained a 163-base pair deletion and encoded a deleted form of FCP1 (FCP1b) that lacks the last 139 amino acids of FCP1a and would, therefore, be unable to interact as strongly with RAP74 (see below). This form of FCP1 would terminate with a unique 62-amino acid sequence read in a different frame and would be comprised of 765 amino acids (see Fig. 1 c). It is probable that the FCP1a and FCP1b mRNAs are produced by the use of alternate splice acceptor sites. The following experiments tested whether FCP1 is related to a previously reported, RAP74-stimulated CTD phosphatase (18Chambers R.S. Dahmus M.E. J. Biol. Chem. 1994; 269: 26243-26248Abstract Full Text PDF PubMed Google Scholar, 19Chambers R.S. Wang B.Q. Burton Z.F. Dahmus M.E. J. Biol. Chem. 1995; 270: 14962-14969Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Fig. 2,a and b, shows that FCP1 immunoreactivity co-purifies precisely with CTD phosphatase activity on phenyl-Superose and Mono Q, two columns that are final steps in the purification of CTD phosphatase (18Chambers R.S. Dahmus M.E. J. Biol. Chem. 1994; 269: 26243-26248Abstract Full Text PDF PubMed Google Scholar). The electrophoretic mobility of this immunoreactive band corresponded to the 150-kDa polypeptide shown previously to co-chromatograph with CTD phosphatase activity (18Chambers R.S. Dahmus M.E. J. Biol. Chem. 1994; 269: 26243-26248Abstract Full Text PDF PubMed Google Scholar). The substrate for the phosphatase assay was 32P-labeled RNAP IIO in which the most carboxyl-terminal serine of the largest RNAP II subunit, IIo, was tagged with 32P. The terminal serine lies outside the consensus CTD repeat and can be selectively phosphorylated with casein kinase II. Dephosphorylation of the CTD by CTD phosphatase results in the removal of unlabeled phosphate within the consensus repeat and an increase in electrophoretic mobility corresponding to that of the unphosphorylated IIa subunit (compare the first two lanes in Fig. 2, a and b). CTD phosphatase was also purified nearly to homogeneity by heparin-Sepharose and phenyl-Sepharose chromatography followed by affinity chromatography on a GST-RAP74-(436–527) column. This preparation contained a major 150-kDa polypeptide (Fig. 3 a, lane 2) that did not bind to a GST control column (lane 1) and trace amounts of other lower molecular weight polypeptides (lane 3). The 150-kDa polypeptide reacted in an immunoblot with affinity purified antibody raised against amino acids 443–669 of recombinant FCP1 (Fig. 3 b, lane 2). This highly purified material also contained CTD phosphatase activity (Fig. 3 d, lanes 10–14). Both affinity purified CTD phosphatase and CTD phosphatase purified by conventional chromatography (Fig. 2, a and b) were strongly stimulated by RAP74 (compare lanes 2 and 3 and lanes 11 and 12 in Fig. 3 d) and had approximately the same specific activities relative to their contents of FCP1. The assay shown in lane 14 contained only phosphorylated RNA polymerase II and affinity purified CTD phosphatase." @default.
• W1996658265 created "2016-06-24" @default.
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• W1996658265 title "FCP1, the RAP74-Interacting Subunit of a Human Protein Phosphatase That Dephosphorylates the Carboxyl-terminal Domain of RNA Polymerase IIO" @default.
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