Reconstitution of Transcription from the Human U6 Small Nuclear RNA Promoter with Eight Recombinant Polypeptides and a Partially Purified RNA Polymerase III Complex
2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês
10.1074/jbc.m100088200
ISSN1083-351X
AutoresSetareh Sepehri Chong, Ping Hu, Nouria Hernandez,
Tópico(s)Fungal and yeast genetics research
ResumoThe human U6 small nuclear (sn) RNA core promoter consists of a proximal sequence element, which recruits the multisubunit factor SNAPc, and a TATA box, which recruits the TATA box-binding protein, TBP. In addition to SNAPc and TBP, transcription from the human U6 promoter requires two well defined factors. The first is hB“, a human homologue of the B” subunit of yeast TFIIIB generally required for transcription of RNA polymerase III genes, and the second is hBRFU, one of two human homologues of the yeast TFIIIB subunit BRF specifically required for transcription of U6-type RNA polymerase III promoters. Here, we have partially purified and characterized a RNA polymerase III complex that can direct transcription from the human U6 promoter when combined with recombinant SNAPc, recombinant TBP, recombinant hB“, and recombinant hBRFU. These results open the way to reconstitution of U6 transcription from entirely defined components. The human U6 small nuclear (sn) RNA core promoter consists of a proximal sequence element, which recruits the multisubunit factor SNAPc, and a TATA box, which recruits the TATA box-binding protein, TBP. In addition to SNAPc and TBP, transcription from the human U6 promoter requires two well defined factors. The first is hB“, a human homologue of the B” subunit of yeast TFIIIB generally required for transcription of RNA polymerase III genes, and the second is hBRFU, one of two human homologues of the yeast TFIIIB subunit BRF specifically required for transcription of U6-type RNA polymerase III promoters. Here, we have partially purified and characterized a RNA polymerase III complex that can direct transcription from the human U6 promoter when combined with recombinant SNAPc, recombinant TBP, recombinant hB“, and recombinant hBRFU. These results open the way to reconstitution of U6 transcription from entirely defined components. TATA box-binding protein recombinant TATA box-binding protein polymerase III The nuclear eucaryotic RNA polymerases cannot recognize their target promoters without the help of transcription factors. In the case of RNA polymerase II, basal transcription from a TATA-containing mRNA promoter can be reconstituted in vitro with a set of recombinant or entirely defined factors, both in Saccharomyces cerevisiae and in mammalian systems (1Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (847) Google Scholar, 2Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 3Myers L.C. Leuther K. Bushnell D.A. Gustafsson C.M. Kornberg R.D. Methods. 1997; 12: 212-216Crossref PubMed Scopus (36) Google Scholar, 4Tirode F. Busso D. Coin F. Egly J.M. Mol. Cell. 1999; 3: 87-95Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In the case of RNA polymerase III, however, this has been achieved only in the yeast system, for transcription of the U6 snRNA gene (5Kassavetis G.A. Nguyen S.T. Kobayashi R. Kumar A. Geiduschek E.P. Pisano M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9786-9790Crossref PubMed Scopus (91) Google Scholar). The yeast U6 snRNA promoter consists of a TATA box located upstream of the transcription start site and A and B boxes located downstream of the transcription start site (6Brow D.A. Guthrie C. Genes Dev. 1990; 4: 1345-1356Crossref PubMed Scopus (108) Google Scholar, 7Eschenlauer J.B. Kaiser M.W. Gerlach V.L. Brow D.A. Mol. Cell. Biol. 1993; 13: 3015-3026Crossref PubMed Scopus (79) Google Scholar). The A and B boxes recruit TFIIIC which, together with the TATA box, then recruit TFIIIB (8Gerlach V.L. Whitehall S.K. Geiduschek E.P. Brow D.A. Mol. Cell. Biol. 1995; 15: 1455-1466Crossref PubMed Google Scholar). In vitro, however, the yeast U6 snRNA promoter can be transcribed in the absence of the B box and TFIIIC because the TATA box is sufficient to recruit TFIIIB (9Moenne A. Camier S. Anderson G. Margottin F. Beggs J. Sentenac A. EMBO J. 1990; 9: 271-277Crossref PubMed Scopus (67) Google Scholar, 10Margottin F. Dujardin G. Gerard M. Egly J.-M. Huet J. Sentenac A. Science. 1991; 251: 424-426Crossref PubMed Scopus (116) Google Scholar). S. cerevisiae TFIIIB is a three-subunit complex consisting of the TATA box-binding protein (TBP)1 (11Kassavetis G.A. Joazeiro C.A.P. Pisano M. Geiduschek P.E. Colbert T. Hahn S. Blanco J.A. Cell. 1992; 71: 1055-1064Abstract Full Text PDF PubMed Scopus (182) Google Scholar), the TFIIB-related factor BRF (TDS4/PCF4) (12Buratowski S. Zhou H. Cell. 1992; 71: 221-230Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 13Colbert T. Hahn S. Genes Dev. 1992; 6: 1940-1949Crossref PubMed Scopus (128) Google Scholar, 14López-De-León A. Librizzi M. Puglia K. Willis I.M. Cell. 1992; 71: 211-220Abstract Full Text PDF PubMed Scopus (108) Google Scholar), and the protein B“ (TFIIIB90/TFC5/TFC7) (5Kassavetis G.A. Nguyen S.T. Kobayashi R. Kumar A. Geiduschek E.P. Pisano M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9786-9790Crossref PubMed Scopus (91) Google Scholar, 15Roberts S. Miller S.J. Lane W.S. Lee S. Hahn S. J. Biol. Chem. 1996; 271: 14903-14909Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 16Rüth J. Conesa C. Dieci G. Lefebvre O. Dusterhoft A. Ottonello S. Sentenac A. EMBO J. 1996; 15: 1941-1949Crossref PubMed Scopus (77) Google Scholar). All three components have been cloned, and TFIIIB has been reconstituted from recombinant subunits (5Kassavetis G.A. Nguyen S.T. Kobayashi R. Kumar A. Geiduschek E.P. Pisano M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9786-9790Crossref PubMed Scopus (91) Google Scholar). Thus, in the case of the yeast U6 promoter, basal RNA polymerase III transcription can be reconstituted in vitro with recombinant TFIIIB and highly purified RNA polymerase III (5Kassavetis G.A. Nguyen S.T. Kobayashi R. Kumar A. Geiduschek E.P. Pisano M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9786-9790Crossref PubMed Scopus (91) Google Scholar). The human U6 snRNA promoter is, unlike the yeast U6 snRNA promoter, entirely located upstream of the transcription start site (see Ref. 17Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholarand references therein). The core promoter consists of a proximal sequence element and a TATA box, and both elements are required for efficient transcription in vitro. The proximal sequence element recruits a multisubunit complex known as SNAPc (18Sadowski C.L. Henry R.W. Lobo S.M. Hernandez N. Genes Dev. 1993; 7: 1535-1548Crossref PubMed Scopus (142) Google Scholar) or PTF (19Murphy S. Yoon J.-B. Gerster T. Roeder R.G. Mol. Cell. Biol. 1992; 12: 3247-3261Crossref PubMed Scopus (150) Google Scholar), which has been reconstituted from recombinant subunits (20Henry R.W. Mittal V. Ma B. Kobayashi R. Hernandez N. Genes Dev. 1998; 12: 2664-2672Crossref PubMed Scopus (65) Google Scholar). The TATA box recruits TBP (21Lobo S.M. Lister J. Sullivan M.L. Hernandez N. Genes Dev. 1991; 5: 1477-1489Crossref PubMed Scopus (99) Google Scholar, 22Simmen K.A. Bernues J. Parry H.D. Stunnenberg H.G. Berkenstam A. Cavallini B. Egly J.-M. Mattaj I.W. EMBO J. 1991; 10: 1853-1862Crossref PubMed Scopus (78) Google Scholar). Until recently, however, little was known about which other TFIIIB components were required for human U6 transcription because mammalian TFIIIB was only partially characterized. We recently isolated cDNAs encoding hB“, a human homologue of yeast B”, and showed that this factor is required for RNA polymerase III transcription from the U6 promoter as well as from gene-internal promoters (23Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar). We also isolated cDNAs encoding hBRFU, a novel homologue of yeast BRF. Unlike hBRF, a previously characterized homologue of yeast BRF that is functional for transcription from gene-internal promoter but not from the U6 promoter, hBRFU is specifically required for U6 transcription (23Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar). Thus, in a extract depleted of both BRF and BRFU, addition of recombinant BRF (and recombinant TBP) specifically reconstituted transcription from the adenovirus 2 VAI gene-internal promoter, but not from the human U6 promoter (17Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar, 24Mital R. Kobayashi R. Hernandez N. Mol. Cell. Biol. 1996; 16: 7031-7042Crossref PubMed Scopus (67) Google Scholar). Reciprocally, addition of recombinant hBRFU reconstituted transcription from the human U6 promoter, but not from the VAI promoter (23Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar). A protein identical to hBRFU (called TFIIIB50) was very recently isolated by Teichmann et al. (25Teichmann M. Wang Z. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14200-14205Crossref PubMed Scopus (68) Google Scholar) as part of a complex containing several other polypeptides. In the transcription system used by these authors, addition of recombinant hBRFU to a depleted fraction did not reconstitute U6 transcription, but addition of a BRFU-containing complex isolated from HeLa cells expressing a tagged BRFU did, suggesting that the factors associated with BFRU were absolutely required for U6 transcription. In addition, a protein called BRF2 and corresponding to a splice variant of hBRF was also reported to be involved in U6 transcription (26McCulloch V. Hardin P. Peng W. Ruppert J.M. Lobo-Ruppert S.M. EMBO J. 2000; 19: 4134-4143Crossref PubMed Scopus (29) Google Scholar). In this case also, addition of recombinant BRF2 could not reconstitute U6 transcription to a depleted extract, but addition of a BRF2-containing complex immunoprecipitated from human culture cells expressing tagged BRF2 could. Here we show that we can purify an RNA polymerase III complex that, together with recombinant SNAPc, TBP, hB“, and hBRFU, can reconstitute transcription from the human U6 promoter. These results confirm the essential role of hB”, and indicate that all factors absolutely required for U6 transcription in vitro are present in the RNA polymerase III complex. Whole cell extract was prepared from HeLa suspension cells as described (27Maroney P.A. Hannon G.J. Nielsen T.W. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 709-713Crossref PubMed Scopus (35) Google Scholar) and dialyzed against buffer D50 (50 mm HEPES, pH 7.9, 0.2 mmEDTA, 20% glycerol, 0.1% Tween 20, 50 mm KCl, 3 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). Glutathione S-transferase-TBP was expressed inEscherichia coli BL21(DE3) cells with the T7 expression system, as previously described (28Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (77) Google Scholar). The protein was then bound to glutathione-agarose beads and TBP was released from the beads by cleavage with thrombin, which cleaved just after the glutathioneS-transferase moiety of the fusion protein. Mono-Q SNAPc was purified as described in Ref. 29Henry R.W. Sadowski C.L. Kobayashi R. Hernandez N. Nature. 1995; 374: 653-657Crossref PubMed Scopus (123) Google Scholar, and recombinant SNAPc was produced in insect cells and purified as described in Ref. 20Henry R.W. Mittal V. Ma B. Kobayashi R. Hernandez N. Genes Dev. 1998; 12: 2664-2672Crossref PubMed Scopus (65) Google Scholar. Recombinant hB“ and hBRFU were produced inE. coli and purified as described (23Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar). For immunoprecipitation of an RNA polymerase III complex, rabbit polyclonal anti-BN51 antibodies (CS682) directed against the last 14 amino acids of BN51 (see Fig. 1), either crude or affinity purified, were cross-linked to protein A-agarose beads (Roche Molecular Biochemicals) with the dimethyl pimelimidate method as described in Ref. 30Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar. The beads were mixed with whole cell extract at a 1:1 ratio, incubated at room temperature for 2 h with agitation, and collected by centrifugation. The beads were then washed 4 times with 120 bead volumes of buffer D100. The washed beads were then tested directly in transcription assays. Alternatively, and with similar results, material bound to the beads was eluted by incubation with 1 bead volume of buffer D100 containing 100 μg/ml of the synthetic peptide against which the antibody was raised, and the eluate was tested in transcription assays. The RNA polymerase III complex active in U6 transcription was purified as follows. 500 ml of HeLa whole cell extract (5785 mg of protein) was first fractionated by an 18–40% ammonium sulfate precipitation. The precipitate was resuspended in 100 ml of HEDPG (25 mmHEPES, pH 7.9, 15% glycerol, 0.1 mm EDTA, 0.1% Tween 20, 3 mm dithiothreitol, 0.5 mmphenylmethylsulfonyl fluoride). The fraction was then diluted with ∼400 ml of HEDPG until the conductivity of the sample was equivalent to that of a 200 mm KCl solution. The sample (1650 mg of protein) was then loaded onto a 240-ml P11 phosphocellulose column (Whatman). The column was washed with 3 column volumes of HEDPG200 (HEDPG with 200 mm KCl) and bound proteins were eluted with a 5-column volume HEDPG gradient extending from 200 to 1000 mm KCl. The P11 fractions containing U6 transcription activity (eluting between 550 and 850 mm KCl) were pooled (126 mg of protein), diluted with 1400 ml of buffer Q (20 mm HEPES, pH 7.9, 5% glycerol, 0.5 mm EDTA, 10 mm MgCl2, 0.1% Tween 20, 3 mmdithiothreitol, and 0.5 mm phenylmethylsulfonyl fluoride) to give a final concentration of 50 mm KCl. The diluted sample was then loaded onto a 10-ml Mono S column (Amersham Pharmacia Biotech). The column was washed with 3 column volumes of Q50 (Q buffer containing 50 mm KCl) and bound proteins were eluted with a 10-column volume Q gradient extending from 50 to 1000 mm KCl. The Mono-S fractions containing the peak of U6 transcription activity (eluted between 200 and 450 mmKCl) were pooled (20 mg of protein) and diluted with buffer Q to a KCl concentration of ∼100 mm. The diluted sample was loaded onto a 1-ml Mono-Q column (Amersham Pharmacia Biotech). The column was washed with 3 column volumes of Q100, and bound proteins were eluted with a 10-column volume Q gradient extending from 100 mm KCl to 600 mm KCl. The peak of U6 transcription activity (3 mg of protein) eluted between 450 and 600 mm KCl. A tenth of the Mono-Q peak (300 μg of protein) was loaded onto a 4.8-ml 10–30% (w/v) sucrose gradient in D100 buffer. The gradient as well as a parallel gradient loaded with protein size markers were then subjected to ultracentrifugation for 11 h at 4 °C, 49,000 rpm, in a SW55 Ti rotor. 100-μl fractions were withdrawn from the top of the gradient, tested for U6 transcription, and fractionated on 5–20% SDS-polyacrylamide gels. The fractions from the reference gradient were analyzed on a 5–20% SDS-polyacrylamide gel. The resolved polypeptides were visualized by staining with silver or Coomassie Brilliant Blue (G-250), or characterized by immunoblotting with antibodies against known proteins. The U6 transcription assays were performed as described (31Lobo S.M. Tanaka M. Sullivan M.L. Hernandez N. Cell. 1992; 71: 1029-1040Abstract Full Text PDF PubMed Scopus (117) Google Scholar) in a total volume of 20 μl with 100 ng of pU6/Hae/RA.2 DNA template. The reactions contained 8 μl of the RNA polymerase III fractions, 10 ng of recombinant TBP, and 4–6 μl of Mono-Q SNAPc. In Fig. 7, the reconstitutions were performed as indicated in the figure legend. We have described before the isolation of a cDNA clone encoding the largest subunit of RNA polymerase III and the generation of an antibody directed against the very C terminus of this subunit (32Sepehri S. Hernandez N. Genome Res. 1997; 7: 1006-1019Crossref PubMed Scopus (23) Google Scholar). To generate a second antibody directed against RNA polymerase III, we took advantage of the published sequence of another RNA polymerase III subunit (33Ittmann M. Ali J. Greco A. Basilico C. Cell Growth Differ. 1993; 4: 503-511PubMed Google Scholar), the BN51 or RPC53 subunit, to generate another anti-peptide antibody. This small subunit which is unique to RNA polymerase III was originally cloned as the gene that complemented a mutation in a temperature-sensitive cell line that arrests in G1 at the non-permissive temperature (33Ittmann M. Ali J. Greco A. Basilico C. Cell Growth Differ. 1993; 4: 503-511PubMed Google Scholar). Since in addition to generating antibodies, we were interested in expressing recombinant BN51 to serve as a marker in immunoblots, we generated a construct for in vitro translation of full-length BN51 from a partial cDNA clone (34Ittmann M. Greco A. Basilico C. Mol. Cell. Biol. 1987; 7: 3386-3393Crossref PubMed Scopus (27) Google Scholar) and from polymerase chain reaction fragments obtained from HeLa cell RNA. The resulting open reading frame (GenBankTMAF346574) differs from the open reading frame predicted by the BN51 sequence deposited in GenBankTM (accession number M17754) by 10 insertions, one deletion, and three nucleotide changes. As a result, the protein sequence predicted by our clone, which we refer to as hRPC53, differs from the original BN51 protein sequence (accession number AAA51838) at several positions within the N-terminal region, as depicted in Fig.1 A. The hRPC53 amino acid sequence is, however, identical to that predicted by a recent entry in GenBankTM (AK026588). We raised rabbit polyclonal antibodies against a peptide corresponding to the last 14 amino acids of the hRPC53 sequence and tested whether this antibody could co-immunoprecipitate hRPC155, the largest subunit of RNA polymerase III. As shown in Fig. 1 B, hRPC155 was detected in HeLa whole cell extract, the starting material for the immunoprecipitation, and in material immunoprecipitated by anti-hRPC53 antibodies, but not in material immunoprecipitated by preimmune antibodies (compare lanes 1 and 3 to lane 2). Thus, the anti-hRPC53 antibodies co-immunoprecipitated the largest subunit of RNA polymerase III, suggesting that they did not disrupt the multisubunit enzyme. Wang and colleagues (35Wang Z. Luo T. Roeder R.G. Genes Dev. 1997; 11: 2371-2382Crossref PubMed Scopus (62) Google Scholar) reported the isolation of an RNA polymerase III holoenzyme capable of directing transcription from RNA polymerase III genes with gene internal promoters. We tested the ability of the RNA polymerase III complex immunoprecipitated with the anti-hRPC53 antibody to direct transcription from the gene external human U6 snRNA promoter, either on its own or combined with recombinant TBP (rTBP), a partially purified SNAPc fraction (Mono-Q SNAPc, (29Henry R.W. Sadowski C.L. Kobayashi R. Hernandez N. Nature. 1995; 374: 653-657Crossref PubMed Scopus (123) Google Scholar), or both. As shown in Fig.2 A, neither rTBP alone, Mono-Q SNAPc alone, nor rTBP together with Mono-Q SNAPc could direct U6 transcription (lanes 1–3). Similarly, immunoprecipitates obtained with preimmune antibodies, or mock reactions performed with just protein A beads, showed no or little activity, with or without added rTBP and Mono-Q SNAPc (lanes 8–15). The anti-hRPC53 immunoprecipitate alone, or complemented with Mono-Q SNAPcor TBP only, showed little or no activity (lanes 4–6). In contrast, the anti-hRPC53 immunoprecipitate complemented with both rTBP and Mono-Q SNAPc resulted in high levels of U6 transcription activity (lane 7). To exclude the possibility that factors required for U6 transcription co-immunoprecipitated with the hRPC53 subunit of RNA polymerase III because of indirect interactions with hRPC53 through bridging DNA rather than because of protein-protein interactions, we performed immunoprecipitations in the presence of the DNA intercalating agent ethidium bromide. This agent can eliminate interactions occurring through bridging DNA (36Lai J.-S. Herr W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6958-6962Crossref PubMed Scopus (397) Google Scholar). As shown in Fig. 2 B, the anti-hRPC53 antibodies immunoprecipitated a complex active in U6 transcription when complemented with Mono-Q SNAPc and rTBP regardless of whether the immunoprecipitation was performed in the absence (lane 3) or presence (lane 5) of ethidium bromide. Together, these results suggest that the anti-hRPC53 antibodies can immunoprecipitate an RNA polymerase III complex that, together with rTBP and biochemically purified Mono-Q SNAPc, is capable of directing accurate and efficient U6 transcription. The results above suggest that an immunoprecipitated RNA polymerase III complex contains all factors required for U6 transcription except for SNAPc, factors other than SNAPc that might be contained in the Mono-Q SNAPc fraction, and TBP (in a form that is functional for U6 transcription). To determine whether we might be able to purify such a complex by another method than immunoprecipitation, we fractionated a HeLa whole cell extract as illustrated in Fig.3. We tested the fractions for their ability to direct U6 transcription when complemented with Mono-Q SNAPc and rTBP, and for the presence of the largest subunit of RNA polymerase III. We first tested various ammonium sulfate precipitation conditions and found that an 18–40% ammonium sulfate cut precipitated the large majority (more than 90%) of the U6 transcription activity, while the majority of the protein (more than 65%) precipitated at ammonium sulfate concentrations higher than 40%. Immunoblots with an anti-hRPC155 antibody showed than the 18–40% ammonium sulfate precipitate also contained more than 90% of the total RNA polymerase III present in the starting extract (not shown). The proteins in the 18–40% ammonium sulfate precipitate were further fractionated on a phosphocellulose column, which was eluted with a linear KCl gradient extending from 200 to 1000 mm KCl. U6 transcription activity eluted in a broad peak between 550 and 850 mm KCl, and as shown in Fig.4, the fractions containing the peak of U6 transcription activity (Fig. 4 A) also contained the peak of RNA polymerase III as measured by the presence of the hRPC155 subunit (Fig. 4 B).Figure 4U6 transcription activity coelutes with the peak of RNA polymerase III on a P11 phosphocellulose column. A, whole cell extract (lane 1), or the flow-through (lane 2) and fractions indicated at the top (lanes 3–16) from an analytical P11 column, or just rTBP and Mono-Q SNAPc (lane 17) were tested for U6 transcription. The reactions in lanes 2–16 were complemented with rTBP and Mono-Q SNAPc. The band labeledIC corresponds to a radiolabeled RNA that was added to the transcription reactions to serve as a control for RNA handling and recovery. B, the 18–40% ammonium sulfate fraction (lane 1), or the flow-through (lane 2) and P11 fractions indicated above the lanes (lanes 3–16) were fractionated on a SDS-polyacrylamide gel and analyzed by immunoblotting with an anti-hRPC155 antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The proteins present in the phosphocellulose peak of activity were further fractionated by successive chromatography on Mono-S and Mono-Q columns, followed by fractionation on a sucrose gradient, as described under “Experimental Procedures.” Fig.5 shows the activity profile of the sucrose gradient fractions. U6 transcription activity was recovered after the 669-kDa size marker in a broad peak with maximum activity in fraction 14. This activity profile coincided with the elution profile of the largest subunit of RNA polymerase III (Fig.6, A and B,panel hRPC155) as well as with that of the hRPC53 subunit of RNA polymerase III (Fig. 6 A), suggesting co-purification with the bulk of RNA polymerase III. Furthermore, the recovery of U6 transcription activity in a single peak after sucrose gradient centrifugation suggested that the activity is contained within a complex, consistent with the observation that it can be immunoprecipitated with antibodies directed against an RNA polymerase III subunit (Fig. 2, above).Figure 6A, the sucrose gradient fractions shown in Fig. 5 were analyzed by immunoblotting with antibodies directed against hRPC155, hRPC53, TBP, and Oct-1, as indicated on theleft. Lane 11 (labeled C) shows 15 μl of Mono-Q peak fraction (the load for the sucrose gradient) except in the Oct-1 panel, where it shows 15 μl of the 18–40% ammonium sulfate fraction. Lane 12 shows 15 μl of the Mono-Q SNAPc fraction. The dots above the lanes indicate the peak of U6 transcription activity. B, the sucrose gradient fractions indicated on top were analyzed by immunoblotting with antibodies directed against hRPC155, hBRF, hB“, La, and TFIIA, as indicated on the left. Lane 11(labeled C) shows 15 μl of the 18–40% ammonium sulfate fraction. Lane 12 shows 15 μl of the Mono-Q SNAPc fraction. The dots above the lanes indicate the peak of U6 transcription activity. C, the Mono-Q SNAPc fraction (lane 2) and whole cell extract (lane 3) were analyzed for the presence of BRFU by immunoblotting with antibodies directed against BRFU. Lane 1shows recombinant BRFU.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We tested the sucrose gradient fractions, as well as the Mono-Q SNAPc fraction used for U6 transcription, for the presence of RNA polymerase III factors, in particular factors that constitute the human TFIIIB activity. The results are shown in Fig. 6. We could detect traces of both TBP and BRF peaking with RNA polymerase III subunits, suggesting association of at least some of this complex with RNA polymerase III. In contrast, hB“, the human homologue of the B” subunit of S. cerevisiae TFIIIB (23Schramm L. Pendergrast P.S. Sun Y. Hernandez N. Genes Dev. 2000; 14: 2650-2663Crossref PubMed Scopus (111) Google Scholar), peaked before the activity, and BRFU, the TFIIB-related factor that functions in U6 transcription, was undetectable in all the sucrose gradient fractions (not shown). It is noteworthy that unlike BRF, both hB“ and hBRFU were present at substantial levels in the Mono-Q SNAPc fraction (Fig. 6, B, lane 12, and C, lane 2), which could thus provide these activities in the transcription reactions in Fig. 5. Both the La protein, which has been implicated in RNA polymerase III transcription termination and in recycling of RNA polymerase III (37Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 851-861Crossref PubMed Scopus (301) Google Scholar, 38Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 841-850Crossref PubMed Scopus (174) Google Scholar, 39Maraia R.J. Kenan D.J. Keene J.D. Mol. Cell. Biol. 1994; 14: 2147-2158Crossref PubMed Scopus (135) Google Scholar, 40Maraia R.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3383-3387Crossref PubMed Scopus (93) Google Scholar, 41Fan H. Sakulich A.L. Goodier J.L. Zhang X. Qin J. Maraia R.J. Cell. 1997; 88: 707-715Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 42Goodier J.L. Fan H. Maraia R.J. Mol. Cell. Biol. 1997; 17: 5823-5832Crossref PubMed Scopus (65) Google Scholar, 43Goodier J.L. Maraia R.J. J. Biol. Chem. 1998; 273: 26110-26116Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), and TFIIA, which has been reported to be required or stimulate RNA polymerase III transcription in a mammalian system (44Waldschmidt R. Seifart K.H. J. Biol. Chem. 1992; 267: 16359-16364Abstract Full Text PDF PubMed Google Scholar,45Meissner W. Holland R. Waldschmidt R. Seifart K.H. Nucleic Acids Res. 1993; 21: 1013-1018Crossref PubMed Scopus (1) Google Scholar), were detectable in the 18–40% ammonium sulfate precipitate but not in the sucrose fractions or the Mono-Q SNAPc fraction (note that the faint band present in all the lanes of the anti-La panel does not co-migrate with the La signal in the 18–40% ammonium sulfate precipitate). Finally, Oct-1, which binds to the octamer sequence in the DSE of RNA polymerase II and RNA polymerase III snRNA promoters and activates snRNA gene transcription, was not detected in the sucrose gradient fractions. Together, these results suggest that U6 transcription activity co-elutes with an RNA polymerase III complex containing RNA polymerase III and a subset of TFIIIB polypeptides, specifically the TBP·BRF complex. We refer to this complex as “sucrose gradient RNA polymerase III” (SG pol III). The SG pol III complex was active when complemented with rTBP and biochemically purified Mono-Q SNAPc. Since we can obtain functionally active recombinant SNAPc (20Henry R.W. Mittal V. Ma B. Kobayashi R. Hernandez N. Genes Dev. 1998; 12: 2664-2672Crossref PubMed Scopus (65) Google Scholar), we asked whether we could replace Mono-Q SNAPc with recombinant SNAPc. We combined a peak sucrose gradient fraction as judged from hRPC155 immunoblots with either rTBP and Mono-Q SNAPc, or rTBP and rSNAPc. Only the combination containing Mono-Q SNAPc resulted in U6 transcription, even though both Mono-Q SNAPc and rSNAPc were active for U6 transcription when tested by complementation of a SNAPc-depleted extract (not shown). We then tested whether we might be able to recover U6 transcription by addition of hB“, hBRFU, or both factors combined. The results are shown in Fig.7. When we combined SG pol III with rTBP and increasing amounts of Mono
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