Interaction between Yeast RNA Polymerase III and Transcription Factor TFIIIC via ABC10α and τ131 Subunits
1999; Elsevier BV; Volume: 274; Issue: 47 Linguagem: Inglês
10.1074/jbc.274.47.33462
ISSN1083-351X
AutoresHélène Dumay‐Odelot, Liudmilla Rubbi, André Sentenac, Christian Marck,
Tópico(s)RNA and protein synthesis mechanisms
ResumoYeast TFIIIC mediates transcription of class III genes by promoting the assembly of a stable TFIIIB-DNA complex that is sufficient for RNA polymerase III recruitment and function. Unexpectedly, we found an interaction in vivo and in vitro between the TFIIIB-recruiting subunit of TFIIIC, τ131, and ABC10α, a small essential subunit common to the three forms of nuclear RNA polymerases. This interaction was mapped to the C-terminal region of ABC10α. A thermosensitive mutation in the C terminus region of ABC10α (rpc10-30) was found to be selectively suppressed by overexpression of a mutant form of τ131 (τ131-ΔTPR2) that lacks the second TPR repeat. Remarkably, therpc10-30 mutation weakened the ABC10α-τ131 interaction, and the suppressive mutation, τ131-ΔTPR2 increased the interaction between the two proteins in the two-hybrid assay. These results point to the potential importance of a functional contact between TFIIIC and RNA polymerase III. Yeast TFIIIC mediates transcription of class III genes by promoting the assembly of a stable TFIIIB-DNA complex that is sufficient for RNA polymerase III recruitment and function. Unexpectedly, we found an interaction in vivo and in vitro between the TFIIIB-recruiting subunit of TFIIIC, τ131, and ABC10α, a small essential subunit common to the three forms of nuclear RNA polymerases. This interaction was mapped to the C-terminal region of ABC10α. A thermosensitive mutation in the C terminus region of ABC10α (rpc10-30) was found to be selectively suppressed by overexpression of a mutant form of τ131 (τ131-ΔTPR2) that lacks the second TPR repeat. Remarkably, therpc10-30 mutation weakened the ABC10α-τ131 interaction, and the suppressive mutation, τ131-ΔTPR2 increased the interaction between the two proteins in the two-hybrid assay. These results point to the potential importance of a functional contact between TFIIIC and RNA polymerase III. polymerase tetratricopeptide repeat yeast-peptone-dextrose open reading frame polyacrylamide gel electrophoresis 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside thermosensitive TATA-binding protein 5-fluoro-orotic acid In eukaryotic cells, RNA polymerase (Pol)1 III is responsible for the transcription of genes encoding tRNAs, 5 S RNA, U6 RNA, and a number of small RNA species. In tRNA genes (tDNA), the internal promoter elements, the A and B blocks, are recognized by TFIIIC. DNA-bound TFIIIC then directs the assembly of TFIIIB that, in turn, is sufficient to recruit RNA polymerase III for multiple transcription cycles. The transcription of all yeast class III genes is a variation of this scheme (1White R.J. RNA Polymerase III Transcription. 2nd Ed. Springer-Verlag/Landes Bioscience, New York1998Crossref Google Scholar). TFIIIC and TFIIIB are multiprotein complexes. Yeast Saccharomyces cerevisiae TFIIIC, also called τ, is a large transcription factor (about 550–600 kDa) that comprises six polypeptides, τ138, τ131, τ95, τ91, τ60, and τ55 (2Gabrielsen O.S. Marzouki N. Ruet A. Sentenac A. Fromageot P. J. Biol. Chem. 1989; 264: 7505-7511Abstract Full Text PDF PubMed Google Scholar, 3Bartholomew B. Kassavetis G.A. Braun B.B. Geiduschek E.P. EMBO J. 1990; 9: 2197-2205Crossref PubMed Scopus (138) Google Scholar, 4Parsons M.C. Weil P.A. J. Biol. Chem. 1990; 265: 5095-5103Abstract Full Text PDF PubMed Google Scholar), that have been characterized by gene cloning and mutagenesis (5Swanson R.N. Conesa C. Lefebvre O. Carles C. Ruet A. Quemeneur E. Gagnon J. Sentenac A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4887-4891Crossref PubMed Scopus (39) Google Scholar, 6Lefebvre O. Carles C. Conesa C. Swanson R.N. Bouet F. Riva M. Sentenac A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10512-10516Crossref PubMed Scopus (37) Google Scholar, 7Parsons M.C. Weil P.A. J. 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EMBO J. 1990; 9: 2197-2205Crossref PubMed Scopus (138) Google Scholar, 12Braun B.R. Bartholomew B. Kassavetis G.A. Geiduschek E.P. J. Mol. Biol. 1992; 228: 1063-1077Crossref PubMed Scopus (73) Google Scholar). The most 3′ subunit, τ91 (12Braun B.R. Bartholomew B. Kassavetis G.A. Geiduschek E.P. J. Mol. Biol. 1992; 228: 1063-1077Crossref PubMed Scopus (73) Google Scholar), participates in DNA binding with τ138 (10Arrebola R. Manaud N. Rozenfeld S. Marsolier M.-C. Lefebvre O. Carles C. Thuriaux P. Conesa C. Sentenac A. Mol. Cell. Biol. 1998; 18: 1-9Crossref PubMed Google Scholar), which is located within and around the B block (3Bartholomew B. Kassavetis G.A. Braun B.B. Geiduschek E.P. EMBO J. 1990; 9: 2197-2205Crossref PubMed Scopus (138) Google Scholar), whereas τ95 and τ55 are accessible to DNA cross-linking within the A block region (3Bartholomew B. Kassavetis G.A. Braun B.B. Geiduschek E.P. EMBO J. 1990; 9: 2197-2205Crossref PubMed Scopus (138) Google Scholar). Finally, the second largest subunit of TFIIIC, τ131, is located the most upstream within the TFIIIB binding region and also extends downstream between the A and B blocks (3Bartholomew B. Kassavetis G.A. Braun B.B. Geiduschek E.P. EMBO J. 1990; 9: 2197-2205Crossref PubMed Scopus (138) Google Scholar). Remarkably, this subunit contains 11 tetratricopeptide repeats (TPR) (8Marck C. Lefebvre O. Carles C. Riva M. Chaussivert N. Ruet A. Sentenac A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4027-4031Crossref PubMed Scopus (54) Google Scholar) known to mediate protein-protein interactions (13Lamb J.R. Tugendreich S. Hieter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (559) Google Scholar). τ131 was shown to interact with two components of TFIIIB, TFIIIB70/BRF1 (14Khoo B. Brophy B. Jackson S.P. Genes Dev. 1994; 8: 2879-2890Crossref PubMed Scopus (108) Google Scholar, 15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and TFIIIB90/B“ (16Rüth J. Conesa C. Dieci G. Lefebvre O. Düsterhöft A. Ottonello S. Sentenac A. EMBO J. 1996; 15: 1941-1949Crossref PubMed Scopus (77) Google Scholar), and the TFIIIB70/BRF1-interacting domain of τ131 was found to lie in the N-terminal region that includes the first TPR unit (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Recently, another subunit of TFIIIC, τ60, was found to participate in TFIIIB recruitment via its interaction with TBP (17Deprez E. Arrebola R. Conesa C. Sentenac A. Mol. Cell. Biol. 1999; (in press)PubMed Google Scholar). S. cerevisiae RNA polymerase (Pol) III is a multisubunit complex comprising 17 polypeptides ranging from 162 to 7.7 kDa (18Chédin S. Ferri M.L. Peyroche G. Andrau J.C. Jourdain S. Lefebvre O. Werner M. Carles C. Sentenac A. Cold Spring Harb. Symp. Quant. Biol. 1998; 63: 381-389Crossref PubMed Scopus (67) Google Scholar), five of which, ABC27, ABC23, ABC14.5, ABC10α, and ABC10β, are shared with Pol I and II. A labile triad of subunits, C34, C31, and C82, has been implicated in the recruitment of Pol III and in transcription initiation (19Werner M. Chaussivert N. Willis I.A. Sentenac A. J. Biol. Chem. 1993; 268: 20721-20724Abstract Full Text PDF PubMed Google Scholar). A mutation in C31 subunit was found to specifically affect transcription initiation but not the catalytic properties of the enzyme (20Thuillier V. Stettler S. Sentenac A. Thuriaux P. Werner M. EMBO J. 1995; 14: 351-359Crossref PubMed Scopus (88) Google Scholar). C34 was found to be localized the furthest upstream on tDNA in initiation complexes (21Bartholomew B. Durkovich D. Kassavetis G.A. Geiduschek E.P. Mol. Cell. Biol. 1993; 13: 942-952Crossref PubMed Scopus (105) Google Scholar, 22Persinger J. Bartholomew G. J. Biol. Chem. 1996; 271: 33039-33046Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), and analysis of mutant Pol III showed that mutations in C34 that decreased its interaction with TFIIIB70/BRF1 affected Pol III recruitment and open complex formation (23Brun I. Sentenac A. Werner M. EMBO J. 1997; 16: 5730-5741Crossref PubMed Scopus (93) Google Scholar). This triad of subunits has its counterpart in human Pol III. These subunits form a subcomplex that is required for transcription initiation (24Wang Z. Roeder R.G. Genes Dev. 1997; 11: 1315-1326Crossref PubMed Scopus (131) Google Scholar). One (hRPC39) of these subunits, homologous to yC34, interacts physically with two components of hTFIIIB (hTBP and hTFIIIB90). More recently, a new essential subunit of yeast Pol III, C17, was also found to interact with C31 and TFIIIB70/BRF1 thus adding a new linkage to the TFIIIB·Pol III connection. 2M. L. Ferri, G. Peyroche, M. Siaut, O. Lefebvre, C. Carles, C. Conesa, and A. Sentenac, submitted for publication. These findings suggest that the recruitment, correct positioning, and activation of Pol III is mediated by multiple contacts between the enzyme and TFIIIB components. In this work we report genetic and biochemical evidence in favor of a direct contact between yeast Pol III and the assembly factor TFIIIC, namely between the common subunit ABC10α and the TFIIIB-assembling subunit of TFIIIC, τ131. Supporting initial two-hybrid experiments, recombinant ABC10α was found to interact in vitro with τ131. A thermosensitive mutation in the conserved C-terminal region of ABC10α, that weakens this interaction, can be rescued by overexpression of a variant form of τ131. These data suggest the existence of functional interactions between TFIIIC and Pol III. The yeast strains used in this study were constructed by genetic techniques based on transformation of lithium acetate-treated cells with standard media and growth conditions (25Scherman F. Methods Enzymol. 1991; 194: 3-20Crossref PubMed Scopus (2557) Google Scholar). Yeast strains are as follows: YLR-01 (Mat a ura3-52 trp1 his3-Δ200 lys2 ade2 ade3Δ rpc10-Δ::HIS3 + pGENs-RPC10) (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar); YLR-06 (Mat a ura3-52 trp1 his3-Δ200 lys2 ade2 ade3Δrpc10-Δ::HIS3 + pGEN-rpc10-30) (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar); YLR-03 (Mat a ura3-52 trp1 his3-Δ200 lys2 ade2 ade3Δrpc10-Δ::HIS3 + pGEN-rpc10-11) (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar); MW670 (Mat a ura3-52 trp1-Δ63 his3-Δ200 lys2-801 ade2-101 leu2-Δ1rpc160-Δ1::HIS3+ pC160-112 TRP1 CEN4rpc160-112) (27Dieci G. Hermann-Le Denmat S. Lukhtanov E. Thuriaux P. Werner M. Sentenac A. EMBO J. 1995; 14: 3766-3776Crossref PubMed Scopus (65) Google Scholar); MW1029 (Mat a ura3-52 trp1-Δ63 his3-Δ200 lys2-801 ade2-101 leu2-Δ1 rpc160-Δ1::HIS3 + pC160-112 TRP1 CEN4 rpc160-270) (20Thuillier V. Stettler S. Sentenac A. Thuriaux P. Werner M. EMBO J. 1995; 14: 351-359Crossref PubMed Scopus (88) Google Scholar); SC91 (Mat α ura3-52 his3-Δ200 lys2-801 ade2-101 leu2-Δ1 rpc53::HIS3–2 TRP1::rpc53(256–424)) (28Chiannilkulchai N. Moenne A. Sentenac A. Mann C. J. Biol. Chem. 1992; 267: 23099-23107Abstract Full Text PDF PubMed Google Scholar); D132-1D (Mat a ura3-52 his3-Δ200 lys2-801 ade2-101 rpc31-236) (20Thuillier V. Stettler S. Sentenac A. Thuriaux P. Werner M. EMBO J. 1995; 14: 351-359Crossref PubMed Scopus (88) Google Scholar). All mutants used in this work have been described previously: τ131-ΔN2, τ131-ΔN3, τ131-ΔN4, τ131-ΔTPR1, τ131-ΔTPR2, τ131-ΔTPR3, τ131-Δbasic2, τ131-Δloop2, τ131-bHLH, τ131–0TPR, τ131–1TPR, τ131–5TPR, and τ131–9TPR (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) (see Fig.1 B); rpc10–14 (E68*), rpc10–15(Q66*), rpc10–16 (L64*) and rpc10-30 (R60YV65D),rpc10–24 (R60E), and rpc10–11(I8NC48R) (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) (see Fig. 3 A).Figure 3Two-hybrid interaction of mutants ABC10α with τ131 or τ131-ΔTPR2. A, the sequences of wild-type (48Carles C. Treich I. Bouet F. Riva M. Sentenac A. J. Biol. Chem. 1991; 266: 24092-24096Abstract Full Text PDF PubMed Google Scholar) and mutant (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) ABC10α proteins are shown; asterisks indicate stop codons.Bold and capitalized letters have the same meaning as in Fig. 5 A. B, the phenotype of the ABC10α mutants is summarized (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) as follows: +, wild type; −, lethal; ts, thermosensitive; Pol III− or Pol−, specific transcription defect in vivo(26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The level of two-hybrid interaction is indicated as in Fig.1 B. C, effect of the ΔTPR2 mutation on the two-hybrid interaction with ABC10α mutants. The strength of two-hybrid interaction between τ131 (TFC4) or mutant τ131-ΔTPR2 with wild-type (RPC10) or mutant ABC10α (rpc10-30 or -11) was evaluated by β-galactosidase dosage; units are expressed in nanomoles of X-gal hydrolyzed per min and per mg of protein. Black bars denote combinations involving τ131 or mutant τ131-ΔTPR2 and ABC10α or rpc10-30 mutant. Positive (TFC4 × BRF1) and negative (RPC82 × RPC10, ΔTPR2 ×RPC82, etc.) controls are shown for comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Two-hybrid system vectors carryingRPC10 mutant alleles were constructed by cloningBamHI-BclI fragments of pGEN-RPC10derivatives (rpc10-14, rpc10-15,rpc10-16, rpc10-30, rpc10-24, andrpc10-11) into pAS-JR (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) for fusion with GAL4 DNA-binding domain (residues 1–147). Correct in frame fusion and similar expression level of fusion proteins were confirmed by sequencing and immunoblotting analysis. Two-hybrid vectors were used to transform Y526 yeast strain. Independent transformants for each combination of plasmids were grown as patches for 2 days at 30 °C on selective solid medium containing 2% raffinose as carbon source. β-Galactosidase activity was revealed by overlaying cells with 10 ml of 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-gal) agar and incubating plates for 24 h at 37 °C or assayed as described previously (19Werner M. Chaussivert N. Willis I.A. Sentenac A. J. Biol. Chem. 1993; 268: 20721-20724Abstract Full Text PDF PubMed Google Scholar). The interaction between TFIIIB70/BRF1 and τ131 was used as a reference (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). pRSETthio/RPC10 (kindly provided by J.-M. Buhler) was generated by subcloning the entire RPC10 coding sequence (obtained by polymerase chain reaction from genomic DNA) in the T7 polymerase expression vector pRSETA (Invitrogen) at a BamHI site. This construct produced a ABC10α-thioredoxin fusion protein, tagged with six histidines and T7-TagTM at the N terminus of rABC10α. Formation of inclusion bodies in the Escherichia coli cytoplasm was prevented by the thioredoxin moiety. E. coli strain BL21(DE3)(pLysS) was transformed with pRSETthio/RPC10, and cultures were grown at 37 °C up to an A 600 of 0.4. Then isopropyl-β-thiogalactopyranoside was added (0.5 mm final concentration), and induced cultures were grown for 2 h at 30 °C. rABC10α was purified under native conditions by chromatography on Ni2+-nitrilotriacetic acid-agarose as specified by the manufacturer (Qiagen) with minor modifications as follows. Bacteria were harvested by centrifugation and resuspended in binding buffer (5 mm imidazole, 500 mm NaCl, 20 mm Tris-HCl, pH 7.9, protease inhibitors (Roche Molecular Biochemicals)) and lysed by heat shock and treatment with lysozyme (0.1 mg/ml final). The lysate was centrifuged, and the protein extract was added to Ni2+-nitrilotriacetic acid-agarose beads equilibrated in the binding buffer. After 1 h at 4 °C, the flow-through fraction was removed, and the resin was washed with binding buffer containing 60 mm imidazole. Bound proteins were eluted stepwise with elution buffer (1m imidazole, 500 mm NaCl, 20 mmTris-HCl, pH 7.5). Samples of eluates were analyzed by Western blotting with anti-T7-TagTM antibodies (Novagen). TheBamHI-BamHI fragment of plasmid pASτ131 (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) was cloned into the pET28c (Novagen) plasmid to produce the wild-type35S-τ131 protein. The BamHI-BamHI fragment of pACTΔTPR2 (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) was cloned into pET28a (Novagen) to produce the mutant 35S-τ131-ΔTPR2 protein (lacking amino acids 162–195) (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). These expression plasmids, pETτ131 and pETΔTPR2, were linearized using AseI and XhoI, respectively. The genes were transcribed and translated in vitro with TNT Coupled Wheat Germ Extract Systems (Promega) in the presence of [35S]methionine. Expression of35S-τ131 (150,000 cpm/ml) and35S-τ131-ΔTPR2 (100,000 cpm/ml) was verified by SDS-PAGE. Partially purified rABC10α-thioredoxin fusion, purified recombinant thioredoxin (Promega), and a control protein extract fromE. coli were subjected to SDS-PAGE and blotted onto nitrocellulose for far Western analysis (29Huet J. Conesa C. Manaud N. Chaussivert N. Sentenac A. Nucleic Acids Res. 1994; 22: 3433-3439Crossref PubMed Scopus (30) Google Scholar). The filter-bound proteins were subjected to a denaturation/renaturation treatment according to the method of Papavassiliou and Bohmann (30Papavassiliou A.G. Bohmann D. Nucleic Acids Res. 1992; 20: 4365-4366Crossref PubMed Scopus (17) Google Scholar). To visualize the binding of 35S-τ131, the 35S-labeled background had to be reduced by addition of 5% low fat milk to the probe. This process was not necessary when probing with35S-τ131-ΔTPR2 due to a stronger interaction of the mutant protein with ABC10α. Full size rABC10α was revealed by anti-T7-TagTM antibodies. Immune complexes were visualized using the ECL™ chemiluminescence kit (Amersham Pharmacia Biotech), and the bound 35S-labeled polypeptides were revealed by autoradiography. The plasmids used for multicopy suppression experiments were constructed as follows: theSalI-XmaI fragments from pCK14 (8Marck C. Lefebvre O. Carles C. Riva M. Chaussivert N. Ruet A. Sentenac A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4027-4031Crossref PubMed Scopus (54) Google Scholar) and pNC14 (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) were cloned into pFL44L to obtain multicopy plasmids bearingTFC4 wild-type gene (pFLτ131) and mutant geneTFC4-ΔTPR2 (pFLΔTPR2) overexpressing τ131 and τ131-ΔTPR2 proteins, respectively. pFL44-RPC10 has been previously described (31Shpakovski G.V. Acker J. Winthzerith M. Lacroix J.F. Thuriaux P. Vigneron M. Mol. Cell. Biol. 1995; 15: 4702-4710Crossref PubMed Scopus (116) Google Scholar). Sequence data for Candida albicans was obtained from the Stanford DNA Sequencing and Technology Center website. Sequencing of C. albicans was accomplished with the support of the NIDR and the Burroughs Wellcome Fund. The sequence of the ABC10α C. albicans ortholog was identified in the unpublished sequence con4–2986 using the NCBI Blast server and the S. cerevisiae sequence as entry. The sequence of ABC10α ortholog in Arabidopsis thaliana has been disclosed using TblastN 2.0 (32Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61050) Google Scholar) run on the NCBI Blast server and non-redundant DNA data base with the human ABC10α sequence as entry. This protein sequence has been tentatively reconstituted from genomic data (2 introns are introduced) (GenBankTM accession numberAB010072). The sequence of P. abyssi was obtained from the Genoscope web site. ABC10α orthologs of Archaeoglobus fulgidus (33Klenk H.-P. Clayton R.A. Tomb J.-F. White O. Nelson K.E. Ketchum K.A. Dodson R.J. Gwinn M. Hickey E.K. Peterson J.D. Nature. 1997; 390: 364-370Crossref PubMed Scopus (1214) Google Scholar), Pyrococcus horikoshii (34Kawarabayasi Y. Sawada M. Horikawa H. Haikawa Y. Hino Y. Yamamoto S. Sekine M. Baba S. Kosugi H. Hosoyama A. DNA Res. 1998; 5: 55-76Crossref PubMed Scopus (557) Google Scholar), P. abyssi, and Methanococcus jannaschii (35Bult C.J. White O. Olsen G.J. Zhou L. Fleischmann R.D. Sutton G.G. Blake J.A. FitzGerald L.M. Clayton R.A. Science. 1996; 273: 1058-1073Crossref PubMed Scopus (2307) Google Scholar) and were disclosed using TblastN 2.0 run on the same server and data base as indicated above. The TPR plots in Fig. 5 display a function that indicates the fit to a TPR consensus sequence matrix extracted from 200 TPR units of S. cerevisiaeproteins. 3H. Dumay and C. Marck, unpublished observations. Peaks are localized at the center of the TPR units. The interaction of τ131 with subunits of the yeast RNA Pol III was explored using the two-hybrid assay. The τ131 gene (TFC4/YGR047c), fused in frame with the GAL4 activation domain was challenged with the complementary fusions of 12 Pol III subunits, C160, C128, C82, C53, AC40, C31, AC19, ABC27, ABC23, ABC14.5, ABC10α, and ABC10β, fused with GAL4 DNA-binding domain. The C34 subunit was not tested since it behaves, by itself, as a strong transcriptional activator (19Werner M. Chaussivert N. Willis I.A. Sentenac A. J. Biol. Chem. 1993; 268: 20721-20724Abstract Full Text PDF PubMed Google Scholar, 36Lalo D. Carles C. Sentenac A. Thuriaux P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5524-5528Crossref PubMed Scopus (111) Google Scholar). The C25 subunit (YKL1/RPC7) (37Sadhale P.P. Woychik N.A. Mol. Cell. Biol. 1994; 14: 6164-6170Crossref PubMed Scopus (44) Google Scholar) was not assayed. Two additional subunits, C17 4M. L. Ferri, personal communication. and C11, 5S. Chédin, personal communication. have been assayed independently and gave a negative two-hybrid interaction with τ131. Of all the Pol III subunits tested with τ131, only ABC10α (RPC10/YHR143wa) (38Treich I. Carles C. Riva M. Sentenac A. Gene Expr. 1992; 2: 31-37PubMed Google Scholar) gave a positive interaction response (Fig.1 A). The β-galactosidase activity level obtained for this interaction was similar to that observed with the τ131-TFIIIB70/BRF1 interaction (Fig. 1 A, see lanes 4 and 6) (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). An interaction was previously noted between C53 and a fragment of τ131 (39Flores A. Briand J.-F. Gadal O. Andrau J.-C. Rubbi L. van Mullem V. Boschiero C. 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 (129) Google Scholar). This interaction could not be detected using the entire τ131 protein. Other components of the Pol III transcription system, TFIIIA, τ138 and TBP, were also tested and gave negative results (not shown). A number of deletion mutants of τ131 were assayed in order to map the interaction domain. As shown in Fig. 1 B, the ABC10α-τ131 interaction could not be restricted to a given subdomain of τ131. Interestingly, however, some deletion mutant forms of τ131, τ131-ΔTPR1, τ131-ΔTPR2, and τ131-ΔTPR3 (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) were found to interact more efficiently with ABC10α than the wild-type τ131 protein. The β-galactosidase activity generated by the τ131-ΔTPR2-ABC10α interaction was increased 3-fold relative to ABC10α-τ131. The interaction of the same collection of τ131 mutants with TFIIIB70/BRF1 (15Chaussivert N. Conesa C. Shaaban S. Sentenac A. J. Biol. Chem. 1995; 270: 15353-15358Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and TFIIIB90/B“ (16Rüth J. Conesa C. Dieci G. Lefebvre O. Düsterhöft A. Ottonello S. Sentenac A. EMBO J. 1996; 15: 1941-1949Crossref PubMed Scopus (77) Google Scholar) has been previously described. The results, summarized in Fig. 1 B,show that the interaction of τ131 variants with the three proteins was quantitatively and qualitatively different. First, the N-terminal part of τ131 interacted specifically with TFIIIB70/BRF1. In contrast, the deletion of the first, second, or third TPR units, which increased the interaction with ABC10α, decreased or did not affect the interaction with TFIIIB70/BRF1. Similarly the ΔTPR1 and ΔTPR3 mutations abrogated and decreased, respectively, the interaction of τ131 with TFIIIB90/B”, whereas the ΔTPR2 mutation strongly stimulated this interaction like in the case of ABC10α. Altogether, these results give weight to the observed ABC10α-τ131 interaction and suggest that a conformational change of τ131 favors this interaction. To confirm the two-hybrid results, a partially purified rABC10α-thioredoxin fusion protein was subjected to SDS-PAGE, transferred to a membrane, denatured, renatured, and probed with35S-τ131 protein and then with antibodies directed to the T7-Tag™ epitope present at the N terminus of rABC10α. As shown in Fig. 2, the 35S-τ131 probe was specifically retained at the level of rABC10α-thioredoxin fusion protein (lane 2) but not by the thioredoxin alone (lane 1). In addition, no signal was observed with a controlE. coli protein extract (lane 3) or when the filter was incubated with another 35S-labeled TFIIIC subunit, τ95, used as a control (data no shown). A similar signal was obtained with the mutant protein 35S-τ131-ΔTPR2 (lanes 4–6). In order to map the domain of ABC10α interacting with τ131, we performed two-hybrid experiments with various mutant proteins (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) (Fig.3, A and B). We first tested three C-terminal deletions removing 3, 5, or 7 amino acids (mutants rpc10-14, rpc10-15, andrpc10-16, respectively) (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). These short deletions were previously shown to confer a lethal phenotype (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The corresponding fusion proteins were normally expressed in vivo suggesting that the lethality did not arise from mutation-induced protein degradation (results not shown). Remarkably, all three deletions were found to abolish the two-hybrid interaction with τ131. Double or single point mutations in the basic C-terminal part of ABC10α (mutants rpc10-30 and rpc10–24) that led to a thermosensitive phenotype (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) also suppressed or weakened the interaction with τ131. On the other hand, a double mutation lying outside this region, rpc10-11, which also caused a thermosensitive phenotype (26Rubbi L. Labarre-Mariotte S. Chédin S. Thuriaux P. J. Biol. Chem. 1999; 274: 31485-31492Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), did not affect the two-hybrid interaction with τ131. These data suggest that τ131 interacts with the C-terminal part of ABC10α and point to a critical role of the conserved Arg-60 residue in this interaction. Interestingly, as shown in Fig. 3 C, the ΔTPR2 mutation increased nearly 3-fold the interaction with the wild-type ABC10α as well as with the two mutant proteins rpc10-11 and rpc10-30. In fact, the decrease of interaction strength caused by the rpc10-30mutation (about 2-fold) was more than compensated by using the ΔTPR2 version of τ131. The
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