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Alternatively spliced hBRF variants function at different RNA polymerase III promoters

2000; Springer Nature; Volume: 19; Issue: 15 Linguagem: Inglês

10.1093/emboj/19.15.4134

1460-2075

Vicki McCulloch, Peter Hardin, Wenchen Peng, J. Michael Ruppert, Susan M. Lobo-Ruppert,

RNA modifications and cancer

Article1 August 2000free access Alternatively spliced hBRF variants function at different RNA polymerase III promoters Vicki McCulloch Vicki McCulloch Departments of Medical Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Peter Hardin Peter Hardin Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Wenchen Peng Wenchen Peng Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author J.Michael Ruppert J.Michael Ruppert Hematology–Oncology, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Susan M. Lobo-Ruppert Corresponding Author Susan M. Lobo-Ruppert Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Vicki McCulloch Vicki McCulloch Departments of Medical Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Peter Hardin Peter Hardin Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Wenchen Peng Wenchen Peng Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author J.Michael Ruppert J.Michael Ruppert Hematology–Oncology, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Susan M. Lobo-Ruppert Corresponding Author Susan M. Lobo-Ruppert Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Author Information Vicki McCulloch1, Peter Hardin2, Wenchen Peng2, J.Michael Ruppert3 and Susan M. Lobo-Ruppert 2 1Departments of Medical Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA 2Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294 USA 3Hematology–Oncology, University of Alabama at Birmingham, Birmingham, AL, 35294 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4134-4143https://doi.org/10.1093/emboj/19.15.4134 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In yeast, a single form of TFIIIB is required for transcription of all RNA polymerase III (pol III) genes. It consists of three subunits: the TATA box-binding protein (TBP), a TFIIB-related factor, BRF, and B″. Human TFIIIB is not as well defined and human pol III promoters differ in their requirements for this activity. A human homolog of yeast BRF was shown to be required for transcription at the gene-internal 5S and VA1 promoters. Whether or not it was also involved in transcription from the gene-external human U6 promoter was unclear. We have isolated cDNAs encoding alternatively spliced forms of human BRF that can complex with TBP. Using immunopurified complexes containing the cloned hBRFs, we show that while hBRF1 functions at the 5S, VA1, 7SL and EBER2 promoters, a different variant, hBRF2, is required at the human U6 promoter. Thus, pol III utilizes different TFIIIB complexes at structurally distinct promoters. Introduction Yeast RNA polymerase III (pol III) is recruited to its cognate promoters primarily via interaction with transcription factor IIIB (TFIIIB) (reviewed in Kumar et al., 1998). Yeast TFIIIB consists of three subunits: the TATA-box binding protein (TBP) (Huet and Sentenac, 1992; Kassavetis et al., 1992), BRF/PCF4/TDS4, a 67 kDa protein related to TFIIB (Buratowski and Zhou, 1992; Colbert and Hahn, 1992; Lopez-De-Leon et al., 1992), and B″/TCF5, a 90 kDa protein (Kassavetis et al., 1995; Roberts et al., 1996; Ruth et al., 1996). All three subunits of TFIIIB are required for transcription of the yeast tRNA, 5S RNA and U6 small nuclear RNA (snRNA) genes (Kassavetis et al., 1990; Huet et al., 1994; Joazeiro et al., 1994); however, these genes differ in the mechanism by which they recruit TFIIIB. TFIIIB cannot bind independently to the 5S or tRNA promoters, but is instead recruited by factors that are already bound to the DNA (Kassavetis et al., 1990). In the case of the 5S promoter, TFIIIA binds the internal control region and is followed by binding of TFIIIC. In contrast, tRNA promoters consist of gene-internal A and B boxes that directly bind TFIIIC. The binding of TFIIIC to the 5S and tRNA promoters is followed by the recruitment of TFIIIB. TFIIIA and TFIIIC can be removed in vitro by treatment with high salt or heparin, leaving TFIIIB stably bound upstream of the transcription initiation site. TFIIIB, in the absence of any other basal factors, can then direct multiple rounds of transcription by pol III from these genes (Kassavetis et al., 1990). A gene-internal A box, a B box in the 3′-flanking sequence and TFIIIC are required for U6 transcription in vivo (Brow and Guthrie, 1990; Eschenlauer et al., 1993). However, the U6 gene also contains an upstream TATA box that can bind TFIIIB directly, so that in vitro the B box and TFIIIC are not required (Joazeiro et al., 1994; Gerlach et al., 1995; Whitehall et al., 1995). The vertebrate pol III transcription apparatus is less well characterized than that of yeast. Vertebrate pol III promoters are more diverse and are classified on the basis of their structures (reviewed in Willis, 1993). Type I genes, exemplified by the 5S RNA gene, contain an internal control region. The presence of gene-internal A and B boxes characterizes the type II tRNA and adenovirus2 VA1 RNA genes. In contrast, the promoters of the type III genes lie entirely upstream of the RNA coding sequences. The basal human U6 promoter, a type III promoter, consists of a TATA box that binds TBP in vitro, and a proximal sequence element (PSE) (Das et al., 1988; Kunkel and Pederson, 1989; Lobo and Hernandez, 1989) that binds the SNAPc/PTF complex (Sadowski et al., 1993; Yoon et al., 1995). TFIIIA and TFIIIC are not required for transcription of the human U6 snRNA gene in vitro (Reddy, 1988; Waldschmidt et al., 1991; Kuhlman et al., 1999). In addition to these three types of genes, there are several that have gene-internal A and B box homologies as well as upstream promoter elements. Among these are the human 7SL gene, the Epstein–Barr virus-encoded EBER1 and EBER2 RNA genes, the Xenopus selenocysteine tRNA gene and the vault RNA gene (reviewed in Willis, 1993). Several lines of evidence suggest that different forms of TFIIIB are required at TATA-less and TATA box-containing promoters. First, human U6 transcription can be reconstituted by the addition of recombinant TBP to a TBP-depleted nuclear extract. In contrast, transcription from the TATA-less VA1, 5S and 7SL promoters can only be restored by the addition of a partially purified 300 kDa TBP-containing complex, 0.38M TFIIIB (Lobo et al., 1992). Secondly, Teichmann and Seifart (1995) identified two forms of TFIIIB, TFIIIB-α and TFIIIB-β, required for transcription of the human U6 and VA1 genes, respectively. Thirdly, Wang and Roeder (1995) isolated a cDNA encoding a 90 kDa human homolog of yeast BRF, TFIIIB90. Like yeast BRF, the N-terminal half of TFIIIB90 is related to the RNA pol II transcription factor TFIIB and contains a Zn2+-binding domain and two imperfect direct repeats. Transcription of the U6 and VA1 genes was abolished in a nuclear extract depleted with polyclonal antibodies against full-length TFIIIB90, but only VA1 transcription could be restored by the addition of recombinant TFIIIB90 and TBP. That U6 transcription was not reconstituted in this way was interpreted to mean that a TFIIIB90-containing complex, distinct from that involved in transcription of the 5S and tRNA genes, was required by U6. Finally, Mital et al. (1996) immunodepleted extracts using antibodies directed against the C-terminal 14 amino acid peptide of TFIIIB90, which they had cloned independently and named hBRF. Removal of >95% of this protein from an extract had no effect on U6 transcription, whereas VA1 transcription was abolished, suggesting that hBRF is not required for U6 transcription. Here we report the cloning of cDNAs encoding variant forms of human BRF that are derived from a single gene by alternative splicing. We refer to the 90 kDa version cloned by Wang and Roeder (1995) and Mital et al. (1996) as hBRF1 and to the other variants as hBRFs 2, 3 and 4. Each variant contains one of the direct repeats found in yeast BRF and hBRF1. We show that all the variants can enter into complexes with TBP and have mapped the domains of each variant that mediate this interaction. We report that hBRF1 is the most active variant in transcription from the 5S, VA1, 7SL and EBER2 promoters, whereas hBRF2 is required for transcription from the U6 promoter. These findings suggest that multiple versions of TFIIIB that differ in composition and function exist in human cells. Results Isolation of cDNAs encoding hBRF variants A human cDNA library (Foster et al., 1999) was screened with a probe derived from hBRF (Mital et al., 1996) and four different hBRF variants were obtained (Figure 1). Partial cDNAs encoding hBRF1 were isolated four times. Two independent isolates of hBRF3 were obtained and one each of hBRFs 2 and 4. Sequences corresponding to the 3′ end of the hBRF4 mRNA are represented in the expressed sequence tag (EST) database (DDBJ/ EMBL/GenBank accession Nos N91109, AI806823 and W20443). Figure 1.Structure of hBRF variants. (A) Schematic representation of variant cDNAs. Translation start (AUG) and stop sites (STOP) are indicated. Repeat 1 and Repeat 2 refer to the regions encoding the imperfect repeats homologous to TFIIB. (B) Amino acid sequences of hBRFs 2, 3 and 4. Arrows indicate the repeat regions. Dashed lines indicate the peptides R2P and hBRF2sp used for raising antibodies. (C) The specificity of anti-peptide antibodies. hBRFs 1–4 were translated in vitro (lanes 1–4) and immunoprecipitated with purified anti-R2P (lanes 5–8) or anti-hBRF2sp antibodies (lanes 9–12). Download figure Download PowerPoint hBRF3 is identical to hBRF1 except that it is N-terminally truncated by the presence of an untranslated 5′ sequence that replaces the region encoding repeat 1 and the Zn2+-binding domain of hBRF1 (Figure 1A). It encodes a 473 amino acid protein (Figure 1B) that migrates at ∼70 kDa on SDS–polyacrylamide gels (Figure 1C, lane 3). The hBRF2 cDNA also contains this 5′-untranslated region, with an additional 500 bp upstream (Figure 1A). Translation of hBRF2, a 139 amino acid protein (Figure 1B) that migrates at ∼23 kDa (Figure 1C, lane 2), initiates at the same methionine as hBRF3. It contains repeat 2 but, after this, a region that is present in hBRFs 1 and 3 is skipped, bringing the sequence downstream of the stop codon in hBRFs 1 and 3 into frame with repeat 2. The hBRF4 cDNA is incomplete and contains part of the Zn2+-binding region, repeat 1 and a region of 86 amino acids that is not homologous to any protein (Figure 1A and B). It encodes a 225 amino acid protein that migrates at ∼25 kDa (Figure 1C, lane 4). The hBRF variants are not library artifacts or products of a gene family as a BAC clone containing part of the hBRF gene, including the unique region of hBRF4, the 5′-untranslated leader of hBRFs 2 and 3 and the regions common to hBRFs 1 and 3, has been sequenced (DDBJ/EMBL/GenBank accession No. AF11170). Furthermore, we have isolated an ∼100 kb human genomic P1 clone that contains the regions unique to all of the variants. The characterization of this clone and the intron–exon structure of the hBRF gene will be described elsewhere (V.McCulloch, J.Grams, W.Peng and S.Lobo-Ruppert, in preparation). Polyclonal antisera were raised against two peptides: R2P, located distal to repeat 2 in hBRFs 1, 2 and 3, and hBRF2sp, an hBRF2-specific peptide (Figure 1B). To confirm the specificity of the antibodies, the hBRFs were translated in vitro (Figure 1C, lanes 1–4) and used in non-denaturing immunoprecipitations (lanes 5–12). Anti-R2P antibodies precipitated the repeat 2-containing hBRFs 1, 2 and 3 (Figure 1C, lanes 5, 6 and 7) but not hBRF4 (lane 8), whereas the anti-hBRF2sp antibodies brought down only hBRF2 (compare lane 10 with lanes 9, 11 and 12). As equal amounts of hBRF2 (five times that shown in the input lane, Figure 1C, lane 2) were used in lanes 10 and 6, the anti-hBRF2sp antibody precipitates hBRF2 less efficiently than does the anti-R2P antibody. The anti-hBRF2sp antibodies do not precipitate or deplete hBRF2 from HeLa nuclear extracts although they detect a protein of the size of hBRF2 in immunoblots of nuclear extracts (Figure 7B, lanes 3 and 4), suggesting that the cognate epitope is masked in the native protein. Figure 2.Characterization of interactions between hBRF variants and TBP. (A) Co-immunoprecipitation of in vitro translated hBRFs with TBP. The hBRF variants, TBP and luc were translated in vitro (lanes 1–6), incubated with TBP in either RIPA buffer (lanes 7–12) or buffer D + 300 mM KCl (lanes 13–18) or without TBP in buffer D + 300 mM KCl (lanes 23–26) and immunoprecipitated with anti-TBP mAb SL30a. (B) Identification of hBRF domains that mediate interactions with TBP. hBRF domains were translated in vitro (lanes 1–6) mixed with TBP and immunoprecipitated with SL30a (lanes 8–14). The different domains are indicated above the lanes: Rep.1, repeat 1; Rep.2, repeat 2; Rep1 + 2, repeat 1 and 2 together; hBRF1,3 sp., C-terminal domain of hBRFs 1 and 3; hBRF2 sp., C-terminal domain unique to hBRF2; hBRF4 sp., C-terminal domain unique to hBRF4. Download figure Download PowerPoint The variant hBRFs form complexes with human TBP in vitro hBRF1 binds TBP in vitro through the TFIIB homology region and, more avidly, through its C-terminal half (Wang and Roeder, 1995; Mital et al., 1996). To determine whether hBRFs 2, 3 or 4 enter into complexes with TBP, they were translated in vitro, mixed with TBP and used in co-immunoprecipitations with the monoclonal antibody (mAb) SL30a, which recognizes human TBP (Ruppert et al., 1996) (Figure 2A). The firefly luciferase (luc) protein was assayed in parallel as a negative control. Ethidium bromide was added to preclude the possibility that DNA might mediate interactions between these proteins (Lai and Herr, 1992). The individually translated input proteins are shown in Figure 2A (lanes 1–6). Under non-denaturing conditions, all of the variants (lanes 14–17), but not luc (lane 13), were brought down with TBP. When 0.5% NP-40 was included in these reactions, only hBRFs 1 and 3 were co-immunoprecipitated with TBP (not shown), indicating that these two variants form stronger complexes with TBP than do hBRFs 2 and 4. When the assay was done in RIPA buffer, the detergents present disrupted protein–protein interactions so that only TBP was immunoprecipitated (Figure 2A, lanes 7–12). The assay was also performed without added TBP, and none of the hBRFs was brought down (Figure 2A, lanes 23–26), confirming that the antibody does not recognize an epitope on any of the variants. Thus, like hBRF1, hBRFs 2, 3 and 4 can enter into complexes with TBP in vitro. Figure 3.Reconstitution of pol III transcription using in vitro translated hBRFs, TBP and phosphocellulose fraction C. (A) Transcription of VA1 and EBER2 genes. Transcription was assayed using fraction C alone (lane 4), C + TBP (lane 1), C + luc (lane 2), luc + TBP (lane 3), the phosphocellulose B and C fractions (lane 5) and increasing amounts of luc (lanes 6–8) or each in vitro translated hBRF (lanes 9–20) with 0.1 μl of TBP and the C fraction. The amounts of hBRFs or luc added (in fmoles) are indicated above the lanes. (B) Graphical representation of the results from (A). Download figure Download PowerPoint To determine which hBRF domains are required for complex formation with TBP (Figure 2B), repeat 1 (lane 2), repeat 2 (lane 3), the two repeats together (lane 4), the C-terminal domain common to hBRFs 1 and 3 (lane 5), and the unique regions of hBRF2 (lane 6) and hBRF4 (lane 7) were translated, mixed with TBP and immunoprecipitated with SL30a (lanes 8–14). Except for the unique region of hBRF2 (Figure 2B, lane 13), all of these truncations (lanes 9–12 and 14) were co-immunoprecipitated detectably with TBP under our conditions. When the assay was performed in the presence of 0.5% NP-40, only the C-terminal region of hBRFs 1 and 3 was brought down with TBP (not shown). This robust interaction may account for the increased strength of association of hBRFs 1 and 3 with TBP. hBRF variants can reconstitute at least basal levels of transcription from various pol III promoters The ability of the hBRF variants to form complexes with TBP raises the possibility that, like hBRF1 and yeast BRF, they may function in transcription by pol III. To examine this possibility, increasing amounts of each variant or the luc control were used in combination with constant amounts of recombinant TBP and the phosphocellulose C fraction to transcribe pol III templates (reconstitution system 1; Figure 3A). In vitro translated hBRF proteins were used, as some of the variants are poorly expressed or insoluble when produced in bacteria. The amounts of hBRFs 2, 3 and 4 used were equalized by phosphoimager analysis. As hBRF1 translates poorly and its transcriptional activity is saturating at lower concentrations, less of this protein (0.3, 0.6 or 0.9 fmol) as compared with the other variants (2, 4 or 6 fmol) was added. Combining phosphocellulose B and C fractions reconstitutes high levels of EBER2 and VA1 transcription (Figure 3A, lane 5) and serves as a positive control. When the B fraction was replaced by hBRF1 and TBP, levels of activity comparable to that of the positive control were obtained from both genes (Figure 3A, compare lanes 9–11 and lane 5). Surprisingly, hBRFs 2, 3 and 4 also stimulate transcription above background levels (Figure 3A, compare lanes 12–14, 15–17, 18–20 and 6–8) but below those obtained with hBRF1 (lanes 9–11). These results are summarized in Figure 3B, in which transcription units (determined by densitometric analysis of the transcripts, with background luc transcription subtracted) are plotted as a function of the amount of hBRF added. Thus, in system 1, activity peaked at 0.6 fmol for hBRF1 whereas addition of higher amounts (4–6 fmol) of the other hBRFs did not stimulate transcription to the same extent as hBRF1. Similar results were obtained with the 7SL gene (J.Grams and V.McCulloch, not shown). Figure 4.Reconstitution of pol III transcription using in vitro translated hBRFs, TBP and anti-R2P antibody-depleted extract. (A) Transcription of the VA1 and U6 genes. VA1 and U6 transcription were assayed in nuclear extract (lane 1), 12CA5 mAb-depleted extract (lane 2), anti-R2P antibody-depleted extract (lane 3) or in anti-R2P antibody-depleted extract complemented with the indicated proteins. The numbers above the lanes indicate the fmoles of luc or hBRF added. (B) Graphical representation of the results from the upper panel in (A). Download figure Download PowerPoint The hBRF variants were also tested in the same way on the human U6 template, but none was able to reconstitute transcription (not shown). As this could be due to a requirement for multiple components of the B fraction, we tried a second approach. HeLa nuclear extracts contain several proteins that are recognized in immunoblots by anti-R2P antibodies (Figure 7B, lane 7), and are removed by depletion with these antibodies (not shown), resulting in inhibition of VA1 and U6 transcription (Figure 4A, compare lane 3 with lane 1 in each panel). Increasing amounts of hBRFs 1, 2 or 3 were then added in combination with TBP to the depleted extract (reconstitution system 2; Figure 4A). In system 2, as in system 1, hBRF1 is the most active variant on the VA1 promoter (Figure 4A, lanes 8–10) while hBRFs 2 and 3 showed lower levels of activity per fmole (lanes 11–16). The results are shown graphically in Figure 4B. However, U6 transcription was not reconstituted in system 2 (Figure 4A, U6 panel, compare lanes 11–22 with lane 1). All possible combinations of the hBRF variants were tested, with additive effects, at best, on VA1 transcription; however, U6 transcription was not reconstituted (not shown). Our results suggest that a modified version of one of the cloned hBRFs, additional components or an unidentified repeat 2-containing hBRF may be required for U6 transcription. Figure 5.Characterization of hBRF complexes assembled in human cells. (A) Detection of purified HA epitope-tagged hBRFs transiently expressed in human cells. Increasing amounts of each hBRF complex were immunoblotted with 12CA5 mAb. Arrows indicate the location of the hBRF variants on the gel. (B) TBP is present in hBRF1 and hBRF3 complexes. hBRF complexes were immunoblotted with anti-TBP mAbs. The light chain of the 12CA5 mAb and TBP are indicated. (C) Cross-linking of TBP to hBRFs expressed in human cells. Extracts from BOSC 23 cells transiently transfected with vector alone (lanes 1 and 2) or HA epitope-tagged hBRF variants (lanes 3–10) were either treated with the cross-linking agent DSP or not (indicated by + or − above the lanes) and immunoprecipitated with 12CA5 mAb under denaturing conditions. The immunoprecipitated proteins were immunoblotted with anti-TBP mAbs. Download figure Download PowerPoint Isolation of complexes containing hBRF variants We next examined the possibility that complexes containing hBRFs 1, 2 or 3 may be involved in U6 transcription. An N-terminally hemagglutinin (HA) epitope-tagged version of each hBRF was introduced into the human embryonal kidney cell line BOSC 23 (Pear et al., 1993) by transient transfection. hBRF complexes were immunopurified from these cells, eluted with HA-peptide and immunoblotted with 12CA5 mAb to verify the expression of each hBRF (Figure 5A). Immunoblotting with anti-R2P antibodies (not shown) detected only the transfected hBRF in each complex, suggesting that multiple repeat 2-containing hBRFs are not present within a single complex. Since hBRF4 is incomplete and we do not have antibodies against it, the possibility that it may co-exist with a repeat 2-containing hBRF in a complex cannot be excluded. To detect associated TBP, hBRF complexes were immunoblotted with a mixture of three anti-TBP mAbs, SL2a, SL26a and SL30a (Ruppert et al., 1996). TBP was co-immunoprecipitated with hBRFs 1 and 3 (Figure 5B, lanes 2 and 4). A background band of the approximate size of TBP is seen in the HA eluates derived from cells transfected with vector alone (Figure 5B, lane 1) or with hBRFs 2 and 4 (lanes 3 and 5). However, if the extracts are treated with the protein cross-linking reagent dithiobis[succinimidylpropionate] (DSP) prior to incubation with 12CA5 mAb, then TBP is brought down with all the hBRFs under denaturing conditions (Figure 5C, lanes 4, 6, 8 and 10). Figure 6.Reconstitution of pol III transcription using hBRF complexes assembled in human cells. (A) Reconstitution of 5S, VA1, 7SL and EBER2 transcription. HeLa nuclear extract (lane 1) was depleted with either 12CA5 mAb (lane 2) or anti-R2P antibodies (lanes 3–15). Increasing amounts of complexes isolated from cells transfected with vector (lanes 4–6) or epitope-tagged hBRFs (lanes 7–15) were added to anti-R2P-depleted nuclear extract. The sizes (in bp) of molecular weight markers are indicated on the right. (B) Reconstitution of VA1 transcription using anti-R2P antibody-depleted extract and hBRF complexes in the presence (lanes 16–28) or absence (lanes 3–15) of exogenous TBP. Reconstitution of U6 transcription in anti-R2P antibody-depleted extracts. In the lower panel, U6 transcription was assayed in nuclear extract (lane 1), depleted extract (lane 2), depleted extract + TBP (lane 3) or with depleted extract, TBP and increasing amounts of the indicated hBRF complexes (lanes 4–15). Download figure Download PowerPoint Reconstitution of transcription from the 5S, 7SL, VA1, EBER2 and U6 promoters using hBRF complexes Complexes containing HA-tagged hBRFs were isolated from transfected cells, immunoblotted with 12CA5 mAb, quantitated by densitometry, and the concentrations of the hBRFs were equalized. Transcription was assayed by adding increasing amounts of each complex to a HeLa nuclear extract depleted with anti-R2P antibodies. This depletion debilitates transcription of the 5S, VA1, 7SL and EBER2 genes (Figure 6A, compare lanes 3 and 1), while depletion with 12CA5 mAb does not (compare lanes 2 and 1). hBRF1-containing complexes restore transcription from these promoters (Figure 6A, lanes 7–9), whereas proteins isolated from mock-transfected cells (lanes 4–6) or complexes that contain hBRFs 2 or 3 (lanes 10–15) do not. Loss of TBP during complex purification does not account for the inability of hBRFs 2 and 3 to reconstitute transcription. hBRF1-containing complexes restore transcription from the VA1 promoter either with or without exogenous TBP (Figure 6B, lanes 7–9 and 20–22, VA1 panel), but exogenous TBP does not allow hBRF2- or hBRF3-containing complexes to support VA1 transcription (Figure 6B, compare lanes 10–15 and 23–28). The addition of luc-programmed reticulocyte lysate, TBP and hBRF complexes to anti-R2P antibody-depleted extract (not shown) gave essentially the same results with VA1 as with the complexes alone. In contrast, when anti-R2P antibody-depleted extract was complemented with in vitro translated hBRFs 2 or 3 and TBP (system 2, Figure 4A) VA1 transcription was restored. A likely explanation is that component(s) present in the hBRF 2 and 3 complexes prevent them from functioning at this promoter. Figure 7.U6, but not VA1, transcription is restored by the addition of recombinant TBP alone to a TBP-depleted nuclear extract. (A) Reconstitution of U6 and VA1 transcription from a TBP-depleted nuclear extract. HeLa nuclear extract was either untreated (lane 1) or immunodepleted with anti-TBP mAbs (lanes 2–7). Recombinant TBP (0.2 and 0.66 fpu; lanes 3 and 4), in vitro translated hBRF1 (3 and 5 μl; lanes 5 and 6) or 0.66 fpu of TBP in combination with 5 μl of hBRF1 (lane 7) were added to the TBP-depleted extract. (B) Anti-TBP mAbs co-deplete hBRF1 but not hBRF2 from nuclear extract. Nuclear extract depleted with anti-TBP mAbs (lanes 2, 4, 6, 8 and 10) or not (lanes 1, 3, 5, 7 and 9) was immunoblotted with pre-immune serum (lanes 1 and 2), anti-hBRF2sp antibodies (lanes 3 and 4), anti-hBRF2sp antibodies + hBRF2sp peptide (lanes 5 and 6), anti-R2P antibodies (lanes 7 and 8) or anti-R2P antibodies + R2P peptide (lanes 9 and 10). Molecular weight markers are indicated on the left. Download figure Download PowerPoint We next assayed the complexes for their ability to restore U6 transcription to an extract depleted with anti-R2P antibodies (Figure 6B, U6 panel). In the absence of exogenous TBP, none of the hBRF complexes restored U6 transcription (not shown). However, when TBP was also added, hBRF2 complexes restored U6 transcription to a level comparable to that of undepleted extract (Figure 6B, compare lanes 10–12 and lane 1), whereas TBP alone (lane 3), or complexes from mock-transfected cells (lanes 4–6) or from cells expressing hBRFs 1 or 3 (lanes 7–9 and lanes 13–15) did not. Exogenous TBP is required because it may be depleted with hBRF2 from the extract by the anti-R2P antibodies, and subsequently lost by washing during purification. These results show that at the VA1, 5S, 7SL and EBER2 promoters, in which gene-internal elements that can potentially bind TFIIIC have been identified (reviewed in Willis et al., 1993), hBRF1 is required for transcription. In contrast, at the human U6 promoter, which does not require TFIIIC (Reddy, 1988; Waldschmidt et al., 1991; Kuhlman et al., 1999), hBRF2 is used preferentially. Anti-TBP antibodies deplete hBRF1–TBP complexes but disrupt hBRF2–TBP complexes TBP alone is sufficient to restore U6 transcription to a nuclear extract immunodepleted of TBP, whereas a TBP-containing fraction, 0.38M TFIIIB, is required to reconstitute transcription from the VA1, 5S and 7SL promoters (Lobo et al., 1992). If hBRF2 does indeed function in U6 transcription, then depletion of an extract with anti-TBP mAbs would be expected to disrupt its interaction with TBP, leaving it behind, whereas hBRF1 would be depleted with TBP. To test this prediction, we depleted a HeLa nuclear extract with a mixture of three anti-TBP mAbs, SL2a, SL33b and SL35a (Ruppert et al., 1996), resulting in reduced U6 and VA1 transcription (Figure 7A, compare lanes 1 and 2). Addition of increasing amounts of recombinant TBP restored U6 but not VA1 transcription (Figure 7A, compare lanes 3 and 4 with lane 2). Addition of hBRF1 (Figure 7A, lanes 5 and 6) had no effect on transcription of either gene, nor did the addition of hBRF1 and TBP increase U6 transcription beyond the level obtained with TBP alone (compare lanes 7 and 4). In contrast, addition of hBRF1 and TBP stimulated VA1 transcription to levels comparable to undepleted extract (Figure 7A, compare lanes 7 and 1), consistent with the results of Mital et al. (1996). The undepleted and depleted extracts were immunoblotted with anti-R2P (Figure 7B, lanes 7 and 8) and anti-hBRF2sp (lanes 3 and 4) anti bodies. A band the size of hBRF1 that is present in the undepleted extract disappears upon deplet