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W2086086978 abstract "Rpb9 is a small subunit of yeast RNA polymerase II participating in elongation and formed of two conserved zinc domains. rpb9 mutants are viable, with a strong sensitivity to nucleotide-depleting drugs. Deleting the C-terminal domain down to the first 57 amino acids has no detectable growth defect. Thus, the critical part of Rpb9 is limited to a N-terminal half that contacts the lobe of the second largest subunit (Rpb2) and forms a β-addition motif with the “jaw” of the largest subunit (Rpb1). Rpb9 has homology to the TFIIS elongation factor, but mutants inactivated for both proteins are indistinguishable fromrpb9 single mutants. In contrast, rpb9 mutants are lethal in cells lacking the histone acetyltransferase activity of the RNA polymerase II Elongator and SAGA factors. In a two-hybrid test, Rpb9 physically interacts with Tfa1, the largest subunit of TFIIE. The interacting fragment, comprising amino acids 62–164 of Tfa1, belongs to a conserved zinc motif. Tfa1 is immunoprecipitated by RNA polymerase II. This co-purification is strongly reduced in rpb9-Δ, suggesting that Rpb9 contributes to the recruitment of TFIIE on RNA polymerase II. Rpb9 is a small subunit of yeast RNA polymerase II participating in elongation and formed of two conserved zinc domains. rpb9 mutants are viable, with a strong sensitivity to nucleotide-depleting drugs. Deleting the C-terminal domain down to the first 57 amino acids has no detectable growth defect. Thus, the critical part of Rpb9 is limited to a N-terminal half that contacts the lobe of the second largest subunit (Rpb2) and forms a β-addition motif with the “jaw” of the largest subunit (Rpb1). Rpb9 has homology to the TFIIS elongation factor, but mutants inactivated for both proteins are indistinguishable fromrpb9 single mutants. In contrast, rpb9 mutants are lethal in cells lacking the histone acetyltransferase activity of the RNA polymerase II Elongator and SAGA factors. In a two-hybrid test, Rpb9 physically interacts with Tfa1, the largest subunit of TFIIE. The interacting fragment, comprising amino acids 62–164 of Tfa1, belongs to a conserved zinc motif. Tfa1 is immunoprecipitated by RNA polymerase II. This co-purification is strongly reduced in rpb9-Δ, suggesting that Rpb9 contributes to the recruitment of TFIIE on RNA polymerase II. polymerase II hemagglutinin general transcription factor IIE Spt-Ada-Gcn5 acetyltransferase The recent determination of the bacterial RNA polymerase (1Zhang G. Campbell E.A. Minakhin L. Richter C. Severinov K. Darst S.A. Cell. 1999; 98: 811-824Abstract Full Text Full Text PDF PubMed Scopus (676) Google Scholar) and yeast RNA polymerase II (Pol II)1 (2Cramer P. Bushnell D.A. Konrberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (985) Google Scholar) core structures has opened a new chapter in transcription studies. The remarkably similar organization of these two enzymes leaves no doubt as to the strong mechanistic conservation of the transcription process. This similarity was anticipated from the sequence homology existing between the β′βα2ω bacterial core enzyme and 5 of the 10 core subunits of Pol II. Rpb1 and Rpb2 are homologues of β′ and β, the Rpb3/Rpb11 heterodimer corresponding to the bacterial α2 dimer, and ω is distantly related to Rpb6 (3Minakhin L. Bhagat S. Brunning A. Campbell E.A. Darst S.A. Ebright R.H. Severinov K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 892-897Crossref PubMed Scopus (174) Google Scholar). On the other hand, Pol II contains five small subunits (Rpb5, Rpb8, Rpb9, Rpb10, Rpb12) not found in the bacterial enzyme. These subunits also belong to the core structure of Pol I and Pol III or are related to it, in the case of Rpb9. Except for Rpb8, they are also akin to archaeal polypeptides (4Langer D. Hain J. Thuriaux P. Zillig W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5768-5772Crossref PubMed Scopus (262) Google Scholar,5Hausner W. Lange U. Musfeldt M. J. Biol. Chem. 2000; 275: 12393-12399Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The present study deals with the Rpb9 subunit, belonging to a conserved family of eukaryotic and archeal zinc-binding polypeptides that also includes the yeast Pol I (Rpa12 (6Nogi Y. Yano R. Dodd J. Carles C. Nomura M. Mol. Cell. Biol. 1993; 13: 114-122Crossref PubMed Scopus (97) Google Scholar)) and Pol III (Rpc11 (7Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (153) Google Scholar)) subunits and the TFIIS elongation factor (8Sawadogo M. Sentenac A. Fromageot P. J. Biol. Chem. 1980; 255: 12-15Abstract Full Text PDF PubMed Google Scholar, 9Awrey D.E. Weibacher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 10Wind M. Reines D. Bioessays. 2000; 22: 327-336Crossref PubMed Scopus (170) Google Scholar) encoded by PPR2 inSaccharomyces cerevisiae (11Hubert J.C. Guyonvarch A. Kammerer B. Exinger F. Liljelund P. Lacroute F. EMBO J. 1983; 2: 2071-2073Crossref PubMed Scopus (50) Google Scholar, 12Nakanishi T. Nakano A. Nomura K. Sekimizu K. Natori S. J. Biol. Chem. 1992; 267: 13200-13204Abstract Full Text PDF PubMed Google Scholar). There is ample evidence that Rpb9 (9Awrey D.E. Weibacher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), Rpc11 (7Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (153) Google Scholar), and TFIIS (9Awrey D.E. Weibacher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13Reines D. Mote Jr., J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1917-1921Crossref PubMed Scopus (93) Google Scholar) control transcription elongation by activating the RNA cleavage activity inherent to all RNA polymerases. The fact that yeast ppr2 (14Exinger F. Lacroute F. Curr. Genet. 1992; 22: 9-11Crossref PubMed Scopus (216) Google Scholar),rpb9 (15Hemming S.A. Jansma D.B. Macgregor P.F. Goryachev A. Friesen J.D. Edwards A.M. J. Biol. Chem. 2000; 275: 35506-35511Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and rpa12 (16Van Mullem V. Landrieux E. Vandenhaute J. Thuriaux P. Mol. Microbiol. 2002; (in press)PubMed Google Scholar) mutants are strongly sensitive to nucleotide-depleting drugs is also consistent with a major elongation defect. Rpb9 is highly conserved in evolution, and the yeast subunit can be replaced in vivo by its human counterpart (17McKune K. Moore P.A. Hull M.W. Woychik N.A. Mol. Cell. Biol. 1995; 15: 6895-6900Crossref PubMed Scopus (60) Google Scholar). However, animal mutants (Drosophila melanogaster) are lethal (18Liu Z. Kontermann R.E. Shulze R.A. Petersen G. Bautz E.K.F. FEBS Lett. 1993; 335: 73-75Crossref PubMed Scopus (2) Google Scholar), whereas yeast null mutants have only a limited growth defect (19Woychik N.A. Lane W.S. Young R.A. J. Biol. Chem. 1991; 266: 19053-19055Abstract Full Text PDF PubMed Google Scholar). Rpa12, Rpb9, and Rpc11 have a related organization in Pol I, Pol II, and Pol III, respectively. These three subunits are essentially made of two zinc-binding domains. Moreover, Rpa12 and Rpb9 hold equivalent positions in the spatial structure of Pol I and Pol II, at the edge of their DNA channels in the “upper” side, which is occupied mainly by the second largest subunit (2Cramer P. Bushnell D.A. Konrberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (985) Google Scholar, 7Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (153) Google Scholar). It was recently shown that the entire C-terminal half of Rpa12 can be removed with no growth defect and without impairing its assembly into Pol I (16Van Mullem V. Landrieux E. Vandenhaute J. Thuriaux P. Mol. Microbiol. 2002; (in press)PubMed Google Scholar, 20Imazawa Y. Imai K. Yao Y. Yamamoto K. Hisatake K. Muramatsu M. Nogi Y. Mol. Gen. Genet. 2001; 264: 852-859Crossref PubMed Scopus (8) Google Scholar). We therefore decided to examine more closely the growth properties of the rpb9 mutant altered in its N-terminal and C-terminal zinc domains and to look for partners of that subunit with other components of the RNA polymerase II transcription complex. Our data revealed a functional connection of Rpb9 with the histone acetyltransferase activity of the Pol II Elongator and SAGA complex and a physical association between Rpb9 and TFIIE. The C-terminal zinc fold of Rpb9 is entirely dispensable in vivo, despite its high sequence conservation. These data will be discussed in the light of models currently proposed for the control of Pol II elongation (21Conaway J.W. Shilatifard A. Dvir A. Conaway R.C. Trends Biochem. Sci. 2000; 25: 375-380Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar,22Gnatt A.L. Cramer P. Fu J. Bushnel D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (755) Google Scholar). Strains CMKy1 (ppr2-Δ::URA3) (23Davie J.K. Kane C.M. Mol. Cell. Biol. 2000; 20: 5960-5973Crossref PubMed Scopus (65) Google Scholar), SL9-6b (rpa12-Δ::LEU2) (16Van Mullem V. Landrieux E. Vandenhaute J. Thuriaux P. Mol. Microbiol. 2002; (in press)PubMed Google Scholar), and WY9 (rpb9-Δ::HIS3) (19Woychik N.A. Lane W.S. Young R.A. J. Biol. Chem. 1991; 266: 19053-19055Abstract Full Text PDF PubMed Google Scholar) have been described. YVV9 (MATα ura3–52 his3-Δ200 leu2 lys2 ade2–1 trp1-Δ63 rpb9-Δ::HIS3) and D386-9b (MATa his3-Δ200 lys2-Δ201 rpb9-Δ::HIS3 trp1-Δ63) were obtained by a WY9 × YPH500 meiotic cross. OG30-4c is a gcn5-Δ::HIS3 mutant, constructed in YPH500 (MATα ura3–52 his3-Δ200 leu2 lys2 ade2–1 trp1-Δ63) (24Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) by replacing the GCN5 coding sequence with a HIS3cassette. 2O. Gadal, unpublished observation. YVV50 (rpb3::3HA) is a YPH500 mutant that encodes a mutant form of the Pol II subunit Rpb3 tagged with a C-terminal hemagglutinin epitope (see Ref. 25Longtine M.S. McKenzie A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philipsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4234) Google Scholar); a meiotic cross with YVV9 yielded the YVV51 (rpb3::3HA rpb9-Δ) segregant. These strains were used to immunopurify Pol II as described previously (16Van Mullem V. Landrieux E. Vandenhaute J. Thuriaux P. Mol. Microbiol. 2002; (in press)PubMed Google Scholar). Western blots were revealed with polyclonal antibodies against Rpb2 (26Riva M. Mémet S. Micouin J.Y. Huet J. Treich I. Dassa J. Young R. Buhler J.M. Sentenac A. Fromageot P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1554-1558Crossref PubMed Scopus (21) Google Scholar) and with mouse monoclonal anti-HA and anti-Myc (Babco). The tfa1 mutants were described previously (27Kuldell N.H. Buratowski S. Mol. Cell. Biol. 1997; 17: 5288-5298Crossref PubMed Scopus (47) Google Scholar). All other yeast strains were from Euroscarf. 3On the Internet at www.uni-frankfurt.de/fb15/mikro/euroscarf/. They are full deletions generated by inserting the KAN-Mx4 cassette in strain BY4741 (MATa his3-Δ1 leu2-Δ0 met15-Δ0 ura3-Δ0). Yeast growth media (16Van Mullem V. Landrieux E. Vandenhaute J. Thuriaux P. Mol. Microbiol. 2002; (in press)PubMed Google Scholar), two-hybrid assays (28Flores A. Briand J.F. Boschiero C. Gadal O. Andrau J.C. Rubbi L. Van Mullem V. Goussot M. Marck C. Carles C. Thuriaux P. Sentenac A. Werner M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7815-7820Crossref PubMed Scopus (128) Google Scholar), and mutagenesis methods (29Briand J.F. Navarro F. Rematier P. Boschiero C. Labarre S. Werner M. Shpakovski G.V. Thuriaux P. Mol. Cell. Biol. 2001; 21: 6056-6065Crossref PubMed Scopus (30) Google Scholar) were as described previously. Plasmids are listed in Table I. pGEN-13MYC was constructed by directional cloning of the 552 ntBamHI-EcoRI fragment from pFA6–13MYC (25Longtine M.S. McKenzie A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philipsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4234) Google Scholar) in the yeast expression vector pGEN (30Shpakovski G.V. Acker J. Wintzerith M. Lacroix J.F. Thuriaux P. Vigneron M. Mol. Cell. Biol. 1995; 15: 4702-4710Crossref PubMed Scopus (116) Google Scholar). pVV101 is a pGEN-13MYC derivative obtained by PCR cloning of the TFA1 coding sequence at theBamHI site upstream of the 13MYC epitope. Plasmids bearing full-length or partially deleted RPB9 alleles (pVV25-pVV111) were generated by PCR amplification of the appropriate oligonucleotides, ending with the BamHI and ClaI sites, and cloned in the centromeric vector pCM185 (31Gari E. Piedrafita L. Aldea M. Herrero E. Yeast. 1997; 13: 837-848Crossref PubMed Scopus (514) Google Scholar). Two-hybrid plasmids were constructed as follows. pGBT9-B9 was obtained by cloning the full-length RPB9 between the BamHI andSalI sites of pGBT9 after PCR amplification. pVV49, pVV50, and pVV51 were made by cloning the corresponding C- and N-terminalrpb9 deletions between the NcoI andBamHI sites of pAS2ΔΔ, and pAD-TFA1 was isolated from a yeast two-hybrid library (32Fromont-Racine M. Rain J.C. Legrain P. Nat. Genet. 1997; 16: 277-282Crossref PubMed Scopus (721) Google Scholar). pVV121-pVV123 were obtained by site-directed mutagenesis of the latter plasmid. All of these constructions were verified by DNA sequencing.Table IPlasmidsPlasmidYeast genesBackbone vector1-aReferences numbers are shown in parentheses.pGBT9-B92μTRP1 GAL4 (1–147)∷RPB9pGBT9 (51)pVV25CEN TRP1ptet07∷RPB9pCM185 (31)pVV26CEN TRP1 ptet07∷rpb9-1,74pCM185pVV35CEN TRP1ptet07∷rpb9-1,70pCM185pVV36CEN TRP1 ptet07∷rpb9-1,66pCM185pVV37CEN TRP1ptet07∷rpb9-1,57pCM185pVV492μTRP1 GAL4 (1–147)∷rpb9-32,122pAS2ΔΔ (28)pVV502μ TRP1 GAL4 (1–147)∷rpb9-57,122pAS2ΔΔpVV512μTRP1 GAL4 (1–147)∷rpb9-1,57pAS2ΔΔpVV111CEN TRP1ptet07∷rpb9–32,122pCM185pVV1122μTRP1 GAL4 (1–147)∷RPA12pAS2ΔΔpAD-Tfa12μ LEU2 GAL4(768–881)∷tfal-62,164pACT2 (32)pVV1212μ LEU2 GAL4(768–881)∷tfa1-62,164 (C127W)pACT2pVV1222μ LEU2 GAL4(768–881)∷tfa1-62,164 (C149S)pACT2pVV1232μ LEU2 GAL4(768–881)∷tfa1-62,164 (C149S)pACT2pGEN-13Myc2μ TRP1 pGK∷13MycpGEN (30)pVV1012μ TRP1 pGK∷tfa1∷13MycpGEN1-a References numbers are shown in parentheses. Open table in a new tab Rpb9 is made of three distinct domains in the spatial structure of Pol II (2Cramer P. Bushnell D.A. Konrberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (985) Google Scholar). As shown in Fig.1 A, each of these domains interacts with a different region of the two large subunits (Rpb1 and Rpb2). The N-terminal zinc fold (Zn1, positions 1–39) contacts the Rpb2 lobe. The β sheet linker (β4, positions 40–52) forms a strong β-addition motif with β28 on the Rpb1 jaw. The C-terminal zinc ribbon (Zn2, positions 53–122) interacts with the Rpb2 funnel (Fig.1 A). An invariant DPTLPR motif on the zinc ribbon has been reported to be critical for the binding of Rpb9 to Pol II in vitro (33Hemming S.A. Edwards A.M. J. Biol. Chem. 2000; 275: 2288-2294Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). rpb9-Δ mutants have a partial growth defect (19Woychik N.A. Lane W.S. Young R.A. J. Biol. Chem. 1991; 266: 19053-19055Abstract Full Text PDF PubMed Google Scholar) and are sensitive to nucleotide-depleting drugs (15Hemming S.A. Jansma D.B. Macgregor P.F. Goryachev A. Friesen J.D. Edwards A.M. J. Biol. Chem. 2000; 275: 35506-35511Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Fig.2 shows that large C-terminal deletions (rpb9-1,57, rpb9-1,66, rpb9-1,70, and rpb9-1,74) are indistinguishable from wild type in term of growth and drug sensitivity. The fact that these deletions always retain the β4 sheet (e.g. rpb9-1,57) but that a more extensive deletion (rpb9-1,47) eliminating the latter region behaves like a null mutant (15Hemming S.A. Jansma D.B. Macgregor P.F. Goryachev A. Friesen J.D. Edwards A.M. J. Biol. Chem. 2000; 275: 35506-35511Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) underscores the importance of the Rpb9-β4/Rpb1-β28 addition motif. In contrast, the DPTLPR motif noted above is deleted in rpb9-1,57 and is thus fully dispensable in vivo. Deleting the N-terminal zinc domain (rpb9-32,122) leads to a null phenotype as already described (15Hemming S.A. Jansma D.B. Macgregor P.F. Goryachev A. Friesen J.D. Edwards A.M. J. Biol. Chem. 2000; 275: 35506-35511Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In the light of the Pol II structure, the main function of Rpb9 may therefore be to hold the Rpb2 lobe (through its interaction with the N-terminal zinc domain of Rpb9) and the Rpb1 jaw (through the β-addition motif mentioned above). As shown Fig. 1 B, the target domain recognized by Rpb9 on the Rpb2 fold is evolutionarily conserved in Pol II. Interestingly, this domain is immediately adjacent to a universally conserved motif shared by all bacterial, archaeal, and eukaryotic enzymes. The mild growth defect of rpb9-Δmutants suggests that other components of the Pol II elongation machinery may be functionally redundant with that subunit. Indeed, the lethality of rpc11 mutants lacking the corresponding Pol III subunit led to the speculation that the latter may combine functions carried separately by Rpb9 and the TFIIS elongation factor in Pol II (7Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (153) Google Scholar). A minor synthetic phenotype was reported betweenppr2-Δ (lacking TFIIS) and rpb9-Δ (15Hemming S.A. Jansma D.B. Macgregor P.F. Goryachev A. Friesen J.D. Edwards A.M. J. Biol. Chem. 2000; 275: 35506-35511Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In our hands, however, rpb9 ppr2-Δ double mutants were indistinguishable from their rpb9-Δ parent (Fig.3 A). Moreover, norpb9 mutant was obtained in a systematic screen based on synthetic lethality with ppr2-Δ (23Davie J.K. Kane C.M. Mol. Cell. Biol. 2000; 20: 5960-5973Crossref PubMed Scopus (65) Google Scholar). The fact thatrpb9-Δ and ppr2-Δ have no additive phenotype implies that the physiological effects of TFIIS on Pol II are mediated by the Rpb9 subunit itself. This is coherent with in vitrodata (9Awrey D.E. Weibacher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) showing that TFIIS promotes read-through and RNA cleavage by Pol II but that this effect is, to a large extent, absent in a mutant form of Pol II lacking the Rpb9 subunit. The Elongator is a transcription factor distinct from TFIIS but also affecting the elongation properties of yeast Pol II. This multisubunit protein is endowed with a histone acetyltransferase activity carried by the Elp3 catalytic subunit (34Wittschieben B.O. Otero G. de Bizemont T. Fellows J. Erdjument-Bromage H. Ohba R. Li Y. Allis C.D. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 4: 123-128Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). elp3-Δ mutants have minor growth defects but are fully lethal when combined with rpb9-Δ (Fig.3 B). elp3-Δ is also lethal withgcn5-Δ deletions of the catalytic subunit of the SAGA complex (35Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (890) Google Scholar). rpb9-Δ itself is synthetic lethal withgcn5-Δ and fails to grow when combined with mutants lacking the Ada2, Ada3, or Spt3 subunits of SAGA (Fig. 3 B).elp3-Δ and rpb9-Δ are both epistatic withsas3-Δ (lacking the histone acetyltransferase component of the Nu3A factor (36John S. Howe L. Tafrov S.T. Grant P.A. Sternglanz R. Workman J.L. Genes Dev. 2000; 14: 1196-1208PubMed Google Scholar) and hpa2-Δ mutants inactivated for the HpaII histone acetyltransferase (Ref. 37Wittschieben B.O. Fellows J. Du W. Stillman D.J. Svejstrup J.Q. EMBO J. 2000; 19: 3060-3068Crossref PubMed Scopus (124) Google Scholar and Fig.3 B)). In summary, our data show a strong synergy between Rpb9, Elp3, and Gcn5, indicating that they are redundant for the same biological function. Deleting the C-terminal domain of Rpb9 (rpb9-1,57) has no detectable effect on gcn5-Δ or elp3-Δ(data not shown). This C-terminal deletion is therefore phenotypically silent by all criteria (growth tests, drug sensitivity, and synthetic lethality) tested so far. The synergy observed here is not a general property of histone acetyltransferases. Intriguingly,gcn5-Δ (but not elp3-Δ) also strongly affects the growth rate of a rpa12-Δ mutant lacking the Pol I counterpart of Rpb9 (Fig. 3 C). This observation buttresses the parallelism already noted between these two subunits. These synthetic lethal effects are not a general consequence of Pol I or Pol II defects because other conditional mutants of Pol I (rpa49-Δ (38Liljelund P. Mariotte M. Buhler J.M. Sentenac A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9302-9305Crossref PubMed Scopus (44) Google Scholar)) or Pol II (rpb1–1 (39Nonet M. Scafe C. Sexton J. Young R. Mol. Cell. Biol. 1987; 7: 1602-1611Crossref PubMed Scopus (270) Google Scholar)) are not at all or are hardly affected in a gcn5-Δ context, as shown Fig. 3 D for rpb1–1. In addition, gcn5-Δ and elp3-Δ are also epistatic with the ppr2-Δ mutation inactivating TFIIS, in keeping with the viability of ppr2-Δ rpb9-Δ double mutants found in this study (data not shown). The data presented above indicate a functional connection between Rpb9 and two chromatin factors associated with RNA Polymerase II but by no means imply a direct physical interaction with these factors. On the other hand, the fact that Rpb9 is located at the outer surface of the spatial Pol II structure (2Cramer P. Bushnell D.A. Konrberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (985) Google Scholar) makes it a likely target of Pol II transcription factors. In an attempt to identify physical partners of Rpb9, we therefore used a GAL4BD::RPB9 fusion as bait in a two-hybrid screening based on a library of yeast genomic fragments (32Fromont-Racine M. Rain J.C. Legrain P. Nat. Genet. 1997; 16: 277-282Crossref PubMed Scopus (721) Google Scholar). Our previous work on other Pol I, Pol II, and Pol III subunits (16Van Mullem V. Landrieux E. Vandenhaute J. Thuriaux P. Mol. Microbiol. 2002; (in press)PubMed Google Scholar, 28Flores A. Briand J.F. Boschiero C. Gadal O. Andrau J.C. Rubbi L. Van Mullem V. Goussot M. Marck C. Carles C. Thuriaux P. Sentenac A. Werner M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7815-7820Crossref PubMed Scopus (128) Google Scholar,29Briand J.F. Navarro F. Rematier P. Boschiero C. Labarre S. Werner M. Shpakovski G.V. Thuriaux P. Mol. Cell. Biol. 2001; 21: 6056-6065Crossref PubMed Scopus (30) Google Scholar) has documented the relevance of this method as a way of mapping interaction domains in the transcription machinery. This two-hybrid approach yielded one interesting candidate that encoded an internal fragment of Tfa1, the largest subunit of the heterodimeric TFIIE transcription factor (Fig.4 A). This interaction is specific of Rpb9 because Tfa1 does not interact with Rpa12 and was never isolated when screening the same library with other RNA polymerase subunits (28Flores A. Briand J.F. Boschiero C. Gadal O. Andrau J.C. Rubbi L. Van Mullem V. Goussot M. Marck C. Carles C. Thuriaux P. Sentenac A. Werner M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7815-7820Crossref PubMed Scopus (128) Google Scholar, 29Briand J.F. Navarro F. Rematier P. Boschiero C. Labarre S. Werner M. Shpakovski G.V. Thuriaux P. Mol. Cell. Biol. 2001; 21: 6056-6065Crossref PubMed Scopus (30) Google Scholar). The two-hybrid response initially obtained with the entire Rpb9 subunit was rather weak, but a strong signal was observed when using an N-terminal deletion removing the first 32 amino acids (GAL4BD::rpb9-33,122). The latter observation implies that the N-terminal zinc domain of Rpb9 is not required for this interaction. However, attempts to more precisely identify which part of Rpb9 is critical have not been conclusive, because neither the N-terminal zinc domain nor the C-terminal zinc ribbon alone were competent for this interaction. Tfa1 itself is evolutionarily conserved on its N-terminal half, whereas the C-terminal half is not, and it can be deleted with only a minor growth defect (27Kuldell N.H. Buratowski S. Mol. Cell. Biol. 1997; 17: 5288-5298Crossref PubMed Scopus (47) Google Scholar, 40Sakurai H. Ohishi T. Fukasawa T. J. Biol. Chem. 1997; 272: 15936-15942Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The fragment recognized by Rpb9 (positions 62–164) corresponds to the conserved part and includes a typical zinc-binding motif. Mutations that are expected to strongly distort that motif such as tfa1-C127W are viable with a conditional growth defect (27Kuldell N.H. Buratowski S. Mol. Cell. Biol. 1997; 17: 5288-5298Crossref PubMed Scopus (47) Google Scholar). They also do not alter the two-hybrid response of the GAL4BD::RPB9 fusion, indicating that the integrity of the zinc motif is probably not required for this interaction (Fig. 4 B). Intriguingly, tfa1-C127Wis strongly sensitive to mycophenolate. Moreover, it is synthetic lethal with rpb9-Δ (Fig. 4, C andD). This lethality is not a general consequence of Pol II-defective mutants, because rpb1-1 mutants (growing slowly and leading to a strong and rapid arrest at 37 °C (39Nonet M. Scafe C. Sexton J. Young R. Mol. Cell. Biol. 1987; 7: 1602-1611Crossref PubMed Scopus (270) Google Scholar)) are not affected by tfa1-C127W (data not shown). Yeast TFIIE directly binds the Pol II core enzyme (41Bushnell D.A. Bamdad C. Kornberg R.D. J. Biol. Chem. 1996; 271: 20170-20174Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) and co-purifies with at least one form of Pol II holoenzyme (42Pan G. Aso T. Greenblatt J. J. Biol. Chem. 1997; 272: 24563-24571Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The two-hybrid interaction found here evidently suggests that TFIIE binding is mediated by Rpb9 via its Tfa1 subunit. To explore this point, we immunopurified Pol II (using a Rpb3::3HA tag) in wild-type and rpb9-Δ strains expressing an epitope-tagged form of Tfa1 (Tfa1::13MYC). Because rpb9-Δ has no effect on the subunit composition of the purified Pol II ((9), differences in the co-purification pattern of Tfa1 can confidently be ascribed to Rpb9 itself. Fig. 5 shows that Tfa1::13MYC co-purifies with the immunoprecipitated Pol II, that rpb9-Δ strongly impairs this co-purification, and that the mutant enzyme nevertheless retains some Tfa1 binding. Taken together, these data strongly suggest that TFIIE binds Pol II by at least two contact points, one of which involves a direct interaction between Rpb9 and the conserved N-terminal part of Tfa1. Rpb9 is formed of two zinc domains that are strongly conserved from yeast to man (17McKune K. Moore P.A. Hull M.W. Woychik N.A. Mol. Cell. Biol. 1995; 15: 6895-6900Crossref PubMed Scopus (60) Google Scholar) and define two separate folds in the spatial structure of Pol II (2Cramer P. Bushnell D.A. Konrberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (985) Google Scholar). In vitro, this subunit controls elongation at the level of the cleaving RNase activity (9Awrey D.E. Weibacher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). This elongation defect is consistent with the strong sensitivity ofrpb9 null mutants to nucleotide-depleting drugs (15Hemming S.A. Jansma D.B. Macgregor P.F. Goryachev A. Friesen J.D. Edwards A.M. J. Biol. Chem. 2000; 275: 35506-35511Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). However, the C-terminal zinc ribbon of Rpb9 can be entirely deleted, down to the first 57 amino acids of Rpb9, without any detectable growth defect and with no increased sensitivity to nucleotide-depleting drugs. Thus, only two elements of Rpb9 are critical for its biological activity, its N-terminal zinc fold (position 1–39), which interacts with the Rpb2 “lobe” in the Pol II spatial structure (2Cramer P. Bushnell D.A. Konrberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (985) Google Scholar), and a β4 “linker” sheet forming a β-addition motif with β28 on the Rpb1 “jaw.” This supports the existence of a mobile lobe-Rpb9-jaw module, as predicted from a comparison of the two structures adopted by Pol II crystals (2Cramer P. Bushnell D.A. Konrberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (985) Google Scholar). The fact that the contact between Rpb9 and the Rpb1 funnel can be disrupted with no detectable effect is surprising given the high conservation of the funnel and that it physically connects Rpb9 to the catalytic pocket of the enzyme. There is a striking parallel with the Rpa12 Pol I subunit in Schizosaccharomyces pombe (20Imazawa Y. Imai K. Yao Y. Yamamoto K. Hisatake K. Muramatsu M. Nogi Y. Mol. Gen. Genet. 2001; 264: 852-859Crossref PubMed Scopus (8) Google Scholar) and inS. cerevisiae (16Van Mullem V. Landrieux E. Vandenhaute J. Thuriaux P. Mol. Microbiol. 2002; (in press)PubMed Google Scholar). In particular, a rpa12deletion retaining the first 60 amino acids of the S. cerevisiae subunit has a wild-type growth and drug sensitivity pattern and suffices to incorporate the mutant subunit into Pol I (16Van Mullem V. Landrieux E. Vandenhaute J. Thuriaux P. Mol. Microbiol. 2002; (in press)PubMed Google Scholar). This parallel is also underscored by the common sensitivity ofrpb9 and rpa12 mutants to nucleotide-depleting agents and by the fact that both are lethal or nearly lethal in agcn5-Δ context (see below). TFIIE is an essential component of Pol II initiation, required during the synthesis of the first few nucleotides and then ejected from the elongating enzyme (43Holstege F.C.P. Tantin D. Carey M. Van der Vliet P.C. Timmers H.T.M. EMBO J. 1995; 14: 810-819Crossref PubMed Scopus (132) Google Scholar, 44Zawel L. Kumar K.P. Reinberg D. Genes Dev. 1995; 9: 1479-1490Crossref PubMed Scopus (266) Google Scholar, 45Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (60) Google Scholar). This factor is known to directly bind Pol II (41Bushnell D.A. Bamdad C. Kornberg R.D. J. Biol. Chem. 1996; 271: 20170-20174Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), and electron microscope crystallography has located this factor in front of the Pol II DNA channel (46Leuther K.K. Bushnell D.A. Kornberg R.D. Cell. 1996; 85: 773-779Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In this study, we found that Rpb9 physically interacted with the large subunit of TFIIE (Tfa1) in a two-hybrid assay, and that this binding involves a conserved domain also present in the human TFIIEα subunit. rpb9−Δmarkedly reduces the co-immunopurification of Tfa1 with Pol II. Moreover, this mutant becomes lethal when combined with a temperature-sensitive allele of Tfa1, tfa1-C127W. These data strongly suggest that the interaction between Rpb9 and Tfa1 is physiologically relevant, allowing Pol II to recruit TFIIE at the edge of the DNA channel. It is tempting to speculate that the association-dissociation of TFIIE is determined by a conformational change in mobile lobe-Rpb9-jaw module hypothesized previously (2Cramer P. Bushnell D.A. Konrberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (985) Google Scholar). RNA polymerases are endowed with an RNA cleavage activity that cleaves the 3′-end of the transcript in halted elongation complexes and presumably determines the processivity of transcription. The catalytic site of cleavage has not been identified. As discussed elsewhere (47Rozenfeld S. Thuriaux P. EMB0 Rep. 2001; 2: 598-603Crossref PubMed Scopus (12) Google Scholar), polymerization and cleavage could be two mutually exclusive forms of the magnesium-binding domain located in the catalytic pocket of Pol II. A Pol III mutant lacking Rpc11 has no cleavage activityin vitro (7Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (153) Google Scholar). TFIIS activates this cleavage activity when recruited on Pol II (13Reines D. Mote Jr., J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1917-1921Crossref PubMed Scopus (93) Google Scholar) in a way that largely depends on the presence of Rpb9 (9Awrey D.E. Weibacher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Unlike rpb9, rpc11 mutants are lethal, which led to the speculation that, in Pol III, Rpc11 may combine functions separately carried by Rpb9 and TFIIS in Pol II (7Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (153) Google Scholar). We show here that the growth defect of rpb9-Δ is not aggravated in ppr2-Δ cells lacking TFIIS. In vivo then, Rpb9 and TFIIS have no additive effects on elongation. Nevertheless, it would be interesting to examine the in vitro properties of the elongating complex in these double mutants. Our data reveal yet another facet of Pol II elongation by showing thatrpb9-Δ cells fail to grow when lacking the histone acetyltransferase activity of either the Elongator (34Wittschieben B.O. Otero G. de Bizemont T. Fellows J. Erdjument-Bromage H. Ohba R. Li Y. Allis C.D. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 4: 123-128Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar) or SAGA complex (35Grant P.A. Duggan L. Cote J. Roberts S.M. Brownell J.E. Candau R. Ohba R. Owen-Hughes T. Allis C.D. Winston F. Berger S.L. Workman J.L. Genes Dev. 1997; 11: 1640-1650Crossref PubMed Scopus (890) Google Scholar). These factors may have partially overlapping functions (37Wittschieben B.O. Fellows J. Du W. Stillman D.J. Svejstrup J.Q. EMBO J. 2000; 19: 3060-3068Crossref PubMed Scopus (124) Google Scholar), but the Elongator is thought to be associated specifically with an elongating form of Pol II (34Wittschieben B.O. Otero G. de Bizemont T. Fellows J. Erdjument-Bromage H. Ohba R. Li Y. Allis C.D. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 4: 123-128Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar). Gcn5, the catalytic subunit of SAGA, acetylates lysine 14 of histone H3 and lysines 8 and 16 of histone H4 (48Kuo M.H. Brownell J.E. Sobel R.E. Ranalli T.A. Cook R.G. Edmondson D.G. Roth S.Y. Allis C.D. Nature. 1996; 383: 269-272Crossref PubMed Scopus (514) Google Scholar). The physiological substrate of the Elongator catalytic subunit, Elp3, is not known (to the best of our knowledge) but may also involve histone tails. Synthetic lethality need not imply a physical interaction between Rpb9 and the Elongator or SAGA factors but clearly means that there is a functional synergy between these proteins. In fact, a simple interpretation of our data is that hypoacetylated histones are an obstacle to elongation, due to the persistence of nucleosomes on the DNA template. It is known that nucleosomal templates are a poor substrate for the elongating enzyme (21Conaway J.W. Shilatifard A. Dvir A. Conaway R.C. Trends Biochem. Sci. 2000; 25: 375-380Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 49Izban M.G. Luse D.S. J. Biol. Chem. 1992; 267: 13647-13655Abstract Full Text PDF PubMed Google Scholar). Rpb9 may thus help the elongating Pol II to overcome these obstacles. This, however, is probably not a direct consequence of the RNA cleaving activity because ppr2-Δ mutants have no effect onelp3-Δ or gcn5-Δ. Rpb9 may be part of a general mechanism allowing Pol II to sense the state of DNA (and, in particular, its degree of nucleosome packaging) prior to elongation (21Conaway J.W. Shilatifard A. Dvir A. Conaway R.C. Trends Biochem. Sci. 2000; 25: 375-380Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) as befits the position of this subunit at the edge of Pol II, ahead of the DNA template (2Cramer P. Bushnell D.A. Konrberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (985) Google Scholar). The related Pol I (Rpa12) (6Nogi Y. Yano R. Dodd J. Carles C. Nomura M. Mol. Cell. Biol. 1993; 13: 114-122Crossref PubMed Scopus (97) Google Scholar, 16Van Mullem V. Landrieux E. Vandenhaute J. Thuriaux P. Mol. Microbiol. 2002; (in press)PubMed Google Scholar) and Pol III (Rpc11) subunits (7Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (153) Google Scholar) probably have a very similar function. Our observation that Rpb9 contributes to the binding of TFIIE onto Pol II also makes sense in this context given the role played by that factor in the transition from initiation to elongation (44Zawel L. Kumar K.P. Reinberg D. Genes Dev. 1995; 9: 1479-1490Crossref PubMed Scopus (266) Google Scholar, 45Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (60) Google Scholar, 50Holstege F.C.P. Fiedler U. Timmers H.T.M. EMBO J. 1997; 16: 7468-7480Crossref PubMed Scopus (159) Google Scholar, 51Bartel P.L. Chien C.T. Sternglanz R. Fields S. BioTechniques. 1993; 14: 920-924PubMed Google Scholar). The molecular basis of this transition is unknown, but it probably requires a major change in Pol II conformation, associated with the opening or closing movements between the upper (Rpb2) and lower (Rpb1) sides of the DNA channel (22Gnatt A.L. Cramer P. Fu J. Bushnel D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (755) Google Scholar). By holding together the lobe of Rpb2 and the jaw of Rpb1, the N-terminal half of Rpb9 could evidently play a major role in these conformational changes. How this ultimately controls the conformation of the catalytic pocket and whether this involves the conserved zinc ribbon of Rpb9 must await further investigations. We are especially grateful to Jean-Christophe Andrau for pointing out to us the existence of a two-hybrid response between Tfa1 and Rpb9. We thank Claire Boschiéro, Olivier Gadal, Sylvie Labarre, and Benoit Van Driessche for their help during this work, Steeve Buratowski for Tfa1 mutants, and Roger Sayre and Christian Marck for the RASMOL and DNA STRIDER software." @default.
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W2086086978 title "The Rpb9 Subunit of RNA Polymerase II Binds Transcription Factor TFIIE and Interferes with the SAGA and Elongator Histone Acetyltransferases" @default.
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