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Functional interchangeability of TFIIIB components from yeast and human cells invitro

1997; Springer Nature; Volume: 16; Issue: 15 Linguagem: Inglês

10.1093/emboj/16.15.4708

1460-2075

Martin Teichmann,

Single-cell and spatial transcriptomics

Article1 August 1997free access Functional interchangeability of TFIIIB components from yeast and human cells in vitro Martin Teichmann Martin Teichmann Institut für Molekularbiologie und Tumorforschung, Lahnstrasse 3, D-35033 Marburg/Lahn, Germany Search for more papers by this author Giorgio Dieci Giorgio Dieci Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette, cedex, France Institut für Molekularbiologie und Tumorforschung, Lahnstrasse 3, D-35033 Marburg/Lahn, Germany Search for more papers by this author Janine Huet Janine Huet Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette, cedex, France Search for more papers by this author Jochen Rüth Jochen Rüth Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette, cedex, France Search for more papers by this author André Sentenac André Sentenac Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette, cedex, France Search for more papers by this author Klaus H Seifart Corresponding Author Klaus H Seifart Institut für Molekularbiologie und Tumorforschung, Lahnstrasse 3, D-35033 Marburg/Lahn, Germany Search for more papers by this author Martin Teichmann Martin Teichmann Institut für Molekularbiologie und Tumorforschung, Lahnstrasse 3, D-35033 Marburg/Lahn, Germany Search for more papers by this author Giorgio Dieci Giorgio Dieci Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette, cedex, France Institut für Molekularbiologie und Tumorforschung, Lahnstrasse 3, D-35033 Marburg/Lahn, Germany Search for more papers by this author Janine Huet Janine Huet Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette, cedex, France Search for more papers by this author Jochen Rüth Jochen Rüth Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette, cedex, France Search for more papers by this author André Sentenac André Sentenac Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette, cedex, France Search for more papers by this author Klaus H Seifart Corresponding Author Klaus H Seifart Institut für Molekularbiologie und Tumorforschung, Lahnstrasse 3, D-35033 Marburg/Lahn, Germany Search for more papers by this author Author Information Martin Teichmann1, Giorgio Dieci2,1,3, Janine Huet2, Jochen Rüth2,4, André Sentenac2 and Klaus H Seifart 1 1Institut für Molekularbiologie und Tumorforschung, Lahnstrasse 3, D-35033 Marburg/Lahn, Germany 2Service de Biochimie et Génétique Moléculaire, Commissariat à l'Energie Atomique-Saclay, F-91191 Gif-sur-Yvette, cedex, France 3Instituto di Scienze Biochimiche, Università di Parma, Viale delle Scienze, I-43100 Parma, Italy 4Institut für Mikrobiologie und Genetik, Universität Wien Biocenter, Dr Bohrgasse 9, A-1030 Wien, österreich The EMBO Journal (1997)16:4708-4716https://doi.org/10.1093/emboj/16.15.4708 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In eukaryotes, TFIIIB is required for proper initiation by RNA polymerase III. In the yeast Saccharomyces cerevisiae a single form of TFIIIB (yTFIIIB) is sufficient for transcription of all pol III genes, whereas in extracts derived from human cells two different hTFIIIB complexes exist which we have previously designated as hTFIIIB-α and hTFIIIB-β. Human TFIIIB-α is a TBP-free entity and must be complemented by TBP for transcription of pol III genes driven by gene external promoters, whereas hTFIIIB-β is a TBP–TAF complex which governs transcription from internal pol III promoters. We show that hTFIIIB-β cannot be replaced by yeast TFIIIB for transcription of tRNA genes, but that the B″ component of yTFIIIB can substitute for hTFIIIB-α activity in transcription of the human U6 gene. Moreover, hTFIIIB-α can be chromatographically divided into activities which are functionally related to yTFIIIE and recombinant yB″90, suggesting that hTFIIIB-α is a human homolog of yeast TFIIIB″. In addition, we show that yeast TBP can only be exchanged against human TBP for in vitro transcription of the human and yeast U6 gene but virtually not for that of the yeast tRNA4Sup gene. This deficiency can be counteracted by a mutant of human TBP (R231K) which is able to replace yeast TBP for transcription of yeast tRNA genes in vitro. Introduction In vertebrate cells, genes expressed by RNA polymerase III (pol III) are controlled either by gene internal class 1 (5S rRNA) or class 2 (e.g. tRNA) promoters or they are governed by class 3 sequence elements located upstream of the initiation site (e.g. U6 snRNA) comprising a TATA-Box, as well as a proximal sequence element (PSE) and a distal sequence element (DSE). In yeast cells, the U6 gene lacks both latter promoter elements. Its expression in vivo depends on a TATA-Box at −25 and a B-Box, which is located downstream of the transcriptional termination site, whereas transcription in vitro minimally requires the TATA-Box. Pol III transcription factors are best characterized in the yeast system, in which case yTFIIIB and yTFIIIC are required together with pol III for the expression of U6 and tRNA genes in vivo (Burnol et al., 1993a, 1993b; Huet et al., 1994; Joazeiro et al., 1994; Kaiser and Brow, 1995; for review, see Willis, 1993; Geiduschek and Kassavetis, 1995). All these yeast factors have been purified to homogeneity and some of them have been cloned. yTFIIIB comprises three polypeptides: TBP, TFIIIB70 and B″90 which are necessary and sufficient for pol III recruitment and accurate initiation (Kassavetis et al., 1995; Roberts et al., 1996). Yet an additional still poorly characterized factor, TFIIIE, appears to be required for efficient transcription (Dieci et al., 1993; Rüth et al., 1996). In human cells, the situation is more complex and class 1, 2 and 3 promoters have individual transcription factor requirements. Class 2 promoters (for example tRNA and VAI gene) depend on TFIIIC2, TFIIIC1, TFIIIB-β and pol III (Yoshinaga et al., 1987; Teichmann and Seifart, 1995 and references therein; Wang and Roeder, 1995, 1996; Yoon et al., 1995). In contrast, class 3 promoters (U6 and 7SK gene) are expressed by PBP (PTF; SNAPc), TFIIIC1, TFIIIB-α and pol III (Waldschmidt et al., 1991; Henry et al., 1995; Teichmann and Seifart, 1995; Wang and Roeder, 1995; Yoon et al., 1995; Oettel et al., 1997). Although a similar function for the yeast and human transcription factors TFIIIC (recognizing the B- and A-Box) and TFIIIB (mediating initiation) have been assumed, there is no sequence similarity between the two human TFIIIC2 subunits cloned thus far and yeast TFIIIC (Swanson et al., 1991; Lefebvre et al., 1992; Parsons and Weil, 1992; Marck et al., 1993; Lagna et al., 1994; L'Etoile et al., 1994; Sinn et al., 1995). Moreover, the human TFIIIB90 protein, although showing significant homology, is only 30% identical in amino acid sequence to its yeast homolog TFIIIB70 (Wang and Roeder, 1995; Mital et al., 1996). In addition, no protein complex has been found which exhibits a PBP- (PTF-, SNAPc-) comparable function in yeast cells. At present, relatively little is known about the functional conservation or divergence of pol III components during evolution. Most studies have concentrated on pol II transcription and many transcription factors have been found to possess sequence similarity and/or a comparable function in yeast and higher eukaryotes (Hoffmann et al., 1990; Pinto et al., 1992; Tschochner et al., 1992; Feaver et al., 1994; Humbert et al., 1994; Verrijzer et al., 1994; Wang et al., 1994; Hisatake et al., 1995; Sun and Hampsey, 1995). Some functional conservation between yeast and human proteins has been shown for the TATA-binding protein in pol II transcription (Cavalini et al., 1988, 1989; Horikoshi et al., 1989; Kelleher et al., 1992). In addition, yeast TBP assembles with human TAFs into a functional TFIID complex when stably transfected into human cells (Zhou et al., 1995) and yeast TBP also has the capacity to replace human TBP in the pol I factor SL1 (Rudloff et al., 1994). Nevertheless, the human TBP gene, introduced into a yeast strain carrying a mutated endogenous TBP, failed to functionally substitute for the Saccharomyces cerevisiae TBP (Cormack et al., 1991; Gill and Tjian, 1991). The region responsible for the species specificity has been mapped to the conserved C-terminus of TBP by these authors. Furthermore, it was shown that simultaneous reintroduction of human TBP and a yeast TBP which was defective for pol I and pol II transcription (Cormack and Struhl, 1993) into the above described mutant yeast strains restored viability. This suggested that the inability of the human TBP to rescue a knock-out yeast TBP strain is due to its inability to support pol III transcription in yeast (Cormack et al., 1994). The interchangeability of yeast and human pol III transcription factors in vitro, which has hitherto not been reported, is not only of fundamental interest from an evolutionary view, but could also help to understand the basic mechanisms underlying the formation of pol III transcription complexes. In this paper we show that human TFIIIB-α activity can be functionally replaced by yeast TFIIIB″ for in vitro transcription of the human U6 gene. All other recombinant or purified pol III-specific factors from both species could neither be exchanged singly nor could they be swapped in pairs. In addition, we present data pointing to a comparable composition of hTFIIIB-α and yeast TFIIIB″ consisting of B″90 and TFIIIE. Furthermore, we demonstrate that human and yeast TBP are completely exchangeable in the transcription of yeast and human U6 genes, whilst only the yeast protein is fully active in the transcription of yeast tRNA genes in vitro. These data provide new insights on the function of TBP in promoter selection and direction of transcription. Results Human TFIIIB90 co-elutes with hTFIIIB-β but not with hTFIIIB-α after chromatography over DEAE Fractogel We could previously show that two different human TFIIIB complexes (hTFIIIB-α and -β) can be chromatographically separated (Teichmann and Seifart, 1995) which are either predominantly active in the transcription of the 5′-regulated human U6 gene (hTFIIIB-α; Figure 1A, lanes 5–7) or which exhibit most of their activity in the expression of RNA polymerase III (pol III) genes governed by internal promoters, exemplified here by transcription of tRNA genes from yeast and human (hTFIIIB-β; Figure 1B, lanes 12–15). As revealed by Western blot analysis, hTFIIIB-β co-elutes with the TATA-binding protein (TBP) and hTFIIIB90 upon chromatography over EMD-DEAE-Fractogel (EDF). In contrast, hTFIIIB-α is free of TBP and hTFIIIB90 (Figure 1C). This finding suggested the possibility that hTFIIIB-α represents a partial human TFIIIB activity. In order to address this question, we attempted to exchange one or both of the human TFIIIB activities against their yeast counterparts. Figure 1.Human TFIIIB-β, but not hTFIIIB-α co-elutes with hTFIIIB90 after chromatography over EMD-DEAE-FRACTOGEL (EDF). (A) Transcription of the human U6 gene (pUhU6wt). In vitro transcription was performed as described in Materials and methods. The position of the U6 transcript is indicated. Three micrograms of pUhU6wt were incubated with the indicated protein fractions. Lane 1: 100 μg PCB; lane 2: 50 μg of EDF flowthrough; lanes 3–17: 50 μg of fractions obtained by elution of the EDF column with a linear gradient from 60 to 450 mM KCl. The fractions assayed for TFIIIB activity were reconstituted with 25 μg TBP-depleted PCC, 10 μg PCA and 25 ng recombinant human TBP. (B) Comparative transcription of the ytRNA3Leu (pUyt-LEU3-0.2) and the human htRNAMet (pUht-MET) gene. In vitro transcription was performed separately for the two genes, as described in Materials and methods. The positions of ytRNA3Leu and htRNAMet transcripts are indicated. Two hundred nanograms of pUyt-LEU3-0.2 or 1 μg of pUht-MET were incubated with the indicated protein fractions. Lane 1: 25 μg HEK-S100; lanes 2 and 4: 20 μg PCB; lanes 3–24: 4.5 μg PCC; lane 5: 10 μg of EDF flowthrough; lanes 6–24: 10 μg of fractions obtained by elution of the EDF column with a linear gradient from 60 to 450 mM KCl. (C) Western-blot analysis using antibodies directed against hTFIIIB90 (polyclonal) or hTBP (monoclonal; Chatterjee et al., 1993). One hundred nanograms of rhTBP (lane 1), 60 μg PCB (lane 2), 30 μg EDF flowthrough (lane 3) and 30 μg of fractions obtained by elution of an EDF column with a linear gradient from 60 to 450 mM KCl (lanes 4–19) were analyzed. SDS–PAGE and Western blot were performed as described in Materials and methods. The positions of hTFIIIB90 and hTBP are indicated. Download figure Download PowerPoint hTFIIIB-α can functionally be replaced by yeast TFIIIB whereas the other pol III transcriptional components are species-specific Yeast TFIIIB (yTFIIIB) is a multiprotein factor that comprises three components: TBP, TFIIIB70 and B″90, which are sufficient to direct accurate transcription of yeast pol III genes in vitro (Kassavetis et al., 1995; Roberts et al., 1996; Rüth et al., 1996). Efficient transcription further requires TFIIIE activity. A partially purified fraction containing yeast TFIIIB was assayed for its ability to replace human TFIIIB-β for in vitro transcription of a human initiator tRNAMet gene and it was found to be insufficient to provide detectable TFIIIB activity (Figure 2A, lanes 3–6). To analyze whether this inability was a consequence of the use of a human instead of a yeast tRNA gene, we expressed the yeast tRNA3Leu gene with purified transcription factors from yeast or human cells, appropriately indicated in Figure 2B. This gene was expressed at approximately equal rates by a complete set of either highly purified human or yeast pol III transcription factors (Figure 2B; compare lanes 6 and 9). However, none of the factors could be exchanged alone, nor could they be swapped in pairs (data not shown). It must be emphasized that the same TFIIIB fraction from yeast cells, which was unable to replace human TFIIIB-β for transcription of the human tRNAMet gene (Figure 2A, lanes 3–6), was capable of efficiently replacing human TFIIIB-α activity, when reconstituted with human transcription factors for human U6 gene transcription (Figure 3A, compare lanes 2–3 and 6–7). The data also show that in vitro transcription of the human U6 gene was not significantly influenced by the DSE, but strongly depended on the presence of the PSE, the removal of which led to a dramatic reduction of transcription, regardless of whether human TFIIIB-α or yeast TFIIIB was used (compare lanes 2–3 and 6–7 of Figure 3A, B and C). Figure 2.Human TFIIIB-β cannot be functionally replaced by Saccharomyces cerevisiae TFIIIB (yTFIIIB). (A) Comparative analysis of hTFIIIB-β or yTFIIIB-containing fractions for transcription of an htRNAMet (pUht-MET) gene. All reactions except lane 7 contained 4.5 μg of PCC from HEK (human embryonal kidney) cells as a minimal reconstitution system to assay TFIIIB activity. Fractions which were assayed for TFIIIB activity were: lane 1: 20 μg PCB; lane 2: none; lanes 3–6: 0.36, 0.72, 1.45 and 2.9 μg of yTFIIIB (Heparin ultrogel 0.26 M ammonium sulfate). Lane 7 had 2.9 μg of yTFIIIB alone. In vitro transcription was performed as described in Materials and methods. The position of htRNAMet transcripts is indicated. (B) In vitro transcription of the ytRNA3Leu gene either with purified human or purified and recombinant yeast transcription factors. Two hundred nanograms of pUyt-Leu3-0.2 were incubated with the indicated protein fractions. Lane 1: 25 μg HEK-S100; lane 6: complete reaction containing 100 ng hTFIIIB-β (mAb-TBP-5M), 900 ng hTFIIIC1 (SO3− 0.6), 300 ng hTFIIIC2 (B-Box-1M), 50 ng hpol III (SO3− 0.55). In lanes 2–5 individual components were deleted as appropriately indicated in Figure 2B. Lane 7: pBR322 DNA-MspI; lane 8: 100 ng ryTFIIIB70, 400 ng yTFIIIB″, 50 ng yTFIIIC, 50 ng ypolC (III); lane 9: the reaction contained the same proteins assayed in lane 8 and was supplemented with 25 ng ryTBP. The position of the ytRNA3Leu transcripts is indicated. In vitro transcription was performed as described in Materials and methods. Download figure Download PowerPoint Figure 3.The B″ component of yeast TFIIIB can functionally replace human TFIIIB-α. (A) Three micrograms of pUhU6wt was incubated with the following protein fractions: lanes 1–13: 25 μg TBP-depleted PCC, 10 μg PCA and 25 ng recombinant human TBP; lane 2: 7.5 μg hTFIIIB-α (SO3− 0.5); lanes 3–5: 15 μg hTFIIIB-α (SO3− 0.5); lane 6: 1.45 μg yeast TFIIIB (Heparin ultrogel 0.26 M ammonium sulfate); lanes 7–9: 2.9 μg yeast TFIIIB; lane 10: 1.3 μg yTFIIIB″ (FT250); lanes 11–13: 2.6 μg yTFIIIB″; lanes 4, 8 and 12: 2 μg/ml α-amanitin; lanes 5, 9 and 13: 300 μg/ml α-amanitin. (B) Three micrograms of pUhU6-0.26 (ΔDSE) were incubated with identical amounts of the protein fractions, depicted in (A). (C) Three micrograms of pUhU6-0.25 (ΔDSEΔPSE) were incubated with identical amounts of the protein fractions, depicted in (A). In vitro transcription was performed as described in Materials and methods. The positions of the U6 transcripts are appropriately indicated in (A), (B) and (C). Download figure Download PowerPoint The B″ component of TFIIIB from Saccharomyces cerevisiae is a functional homolog of human TFIIIB-α In order to further differentiate which of the yeast TFIIIB components could functionally replace human TFIIIB-α for transcription of the human U6 gene we analyzed a partially purified yeast TFIIIB″ (yB″) fraction (Huet et al., 1994) for in vitro exchange experiments. We initially concentrated on this fraction because hTFIIIB-α did not contain hTBP or hTFIIIB90 (Figure 1C) and it was hence expected to functionally resemble yTFIIIB″. As shown in Figure 3A (lanes 10–11), the yB″ fraction alone was sufficient to completely replace hTFIIIB-α activity (Figure 3A, lanes 2–3) and addition of recombinant yeast TFIIIB70 did not further enhance this activity (data not shown). Deletion of upstream promoter elements (DSE and PSE) did not influence the capacity of yB″ to functionally replace human TFIIIB-α for transcription of the human U6 gene, although the promoter strength was dramatically reduced after deletion of the PSE (Figure 3A, B and C; compare lanes 2–3 with lanes 10–11). Transcription of the human U6 gene and its promoter deletion constructs was catalyzed by human RNA polymerase III, independently of whether human TFIIIB-α or yeast TFIIIB components were used, since transcription was insensitive to 2 μg/ml but completely abolished by 300 μg/ml α-amanitin (compare lanes 4–5, 8–9 and 12–13 of Figure 3A, B and C). Human TFIIIB-αis minimally composed of two activities functionally related to TFIIIE and B″90 from yeast cells Since optimal yeast TFIIIB″ (yB″) activity is composed of yTFIIIE and cloned yB″90 (Rüth et al., 1996) and given that yB″ can replace hTFIIIB-α for in vitro transcription of the human U6 gene, we wanted to know whether hTFIIIB-α shows a comparable composition to that of the yeast B″ activity. For this purpose we raised polyclonal antibodies against recombinant yeast B″90 and coupled them covalently to protein A–Sepharose (pAb-B″90–Sepharose). An EMD-SO3−-Fractogel 0.5 fraction containing human TFIIIB-α activity was purified over such a column. Neither the flowthrough nor the fraction eluted with 5 M urea alone were able to fully restore a TFIIIB-α activity comparable to that of the load (Figure 4A, compare lanes 4 and 5–7 respectively, with lane 3). Only the combination of these two fractions yielded an activity comparable with that of loaded hTFIIIB-α (Figure 4A, lanes 8–10). Similarly, recombinant yeast B″90 alone was not able to completely replace hTFIIIB-α in a reconstituted in vitro transcription assay with human transcription factors (Figure 4A, lanes 11–16), but when complemented with the flowthrough of the pAb-B″90–Sepharose a significant hTFIIIB-α-like activity was restored (Figure 4A, lanes 17–22). In addition, partially purified yeast TFIIIE, complemented with the human pAb-B″90–Sepharose 5 M urea fraction, yielded a hybrid TFIIIB activity which to some extent replaced hTFIIIB-α for transcription of the human U6 gene in vitro (Figure 4B, lanes 6–7). Figure 4.Human TFIIIB-α is composed of at least two activities, functionally related to ryB″90 and yTFIIIE. (A) All reactions except in lane 2 were reconstituted with 25 μg TBP-depleted PCC, 10 μg PCA and 25 ng recombinant human TBP. The following fractions were analyzed for their TFIIIB activity: lanes 2 and 3: 15 μg SO3− 0.5 (hTFIIIB-α); lanes 4, 8–10 and 17–22: 15 μg flowthrough of the pAb-B″90–Sepharose; lanes 5–7 and 8–10: 1, 2 and 4 ng of the fraction eluted with 5 M urea from the pAb-B″90–Sepharose; lanes 11–16 and 17–22: 0.5, 1, 2, 4, 8 and 16 ng of Heparin-Fractogel 0.45 M KCl (ryB″90). (B) All reactions were reconstituted with 25 μg TBP-depleted PCC, 10 μg PCA and 25 ng recombinant human TBP. The following fractions were analyzed for their TFIIIB activity: lane 1: 400 ng B″90; lane 2: 2 μg yTFIIIE; lanes 3 and 6–7: 4 μg yTFIIIE; lanes 4–5 and 6–7: 1 and 2 ng of the fraction eluted with 5 M urea from the pAb-B″90–Sepharose. In vitro transcription was performed as described in Materials and methods. The position of the hU6 transcript is indicated. (C) SDS–12.5% PAGE of hTFIIIB-α purified over pAb-B″90–Sepharose. Lane 1: the migration of 0.5 μg of marker proteins is appropriately indicated; lane 2: 50 μg SO3− 0.5 (hTFIIIB-α); lane 3: 50 μg of flowthrough from the pAb-B″90–Sepharose eluting with 60 mM KCl; lane 4: 25 ng of the fraction eluting with 5 M urea from the pAb-B″90–Sepharose. SDS–PAGE was performed as described in Materials and methods. Download figure Download PowerPoint The proteins retained on the pAb-B″90–Sepharose were analyzed by SDS–12.5%-PAGE. The bulk of protein did not bind to the column and hence eluted with the flowthrough (Figure 4C, lane 3) whereas proteins of 90, 67 and 65 kDa were retained by and were eluted with 5 M urea from this column (Figure 4C, lane 4). This procedure led to a significant degree of purification and 1–4 ng of the protein depicted in Figure 4C, lane 4 were highly active in reconstitution of human U6 gene transcription (Figure 4A, lanes 8–10). The polypeptide of ∼90 kDa was not identical to hTFIIIB90 since the latter component was not detectable by Western blot analysis in hTFIIIB-α fractions (Figure 1C). Human and yeast TBP are interchangeable for transcription of human and yeast U6 genes in vitro The TATA-binding protein (TBP) is a component of human as well as yeast TFIIIB. hTFIIIB-α and yeast B″ can be easily separated from TBP employing native chromatographic procedures (Huet et al., 1994; Teichmann and Seifart, 1995). We used TFIIIB activities from both species which were TBP-free and which could be complemented by addition of either human or yeast TBP. As shown in Figure 5A, recombinant TBP from both species was comparably active in the transcription of the human U6 gene in vitro. Figure 5.Recombinant TBP from yeast and human cells is functionally exchangeable for transcription of yeast and human U6 genes but not for transcription of yeast tRNA genes in vitro. (A) Twenty-five micrograms of TBP-depleted PCC, 10 μg PCA and 100 μg TBP-depleted PCB in lanes 1–12 were supplemented with 2.5, 5, 10, 20, 40 and 80 ng of recombinant TBP from human cells (lanes 1–6) or yeast cells (lanes 7–12). In vitro transcription was performed as described in Materials and methods using 3 μg of pUhU6wt as template. (B) Transcription of the yU6 gene. The following protein fractions were incubated with 200 ng pTaq6: Lanes 1 and 3: 100 ng ryTFIIIB70, 25 ng ryTBP and 400 ng yTFIIIB″. Lane 3 additionally contained 50 ng ypolC (III) which was also used to reconstitute the reactions in lanes 2, 4 and 5. The reactions in lanes 4 and 5 contained 100 ng ryTFIIIB70 and 400 ng yTFIIIB″, but ryTBP was omitted. Lanes 2 and 5 contained 25 ng recombinant human TBP. In vitro transcription was performed as described in Materials and methods and the position of the yeast U6 transcript is indicated. (C) Two hundred nanograms of pRS316 (SUP4 ytRNA gene) were incubated with the following protein fractions: 100 ng ryTFIIIB70, 400 ng yTFIIIB″, 50 ng yTFIIIC and 50 ng ypolC (III) in all reactions. Lane 1: 25 ng ryTBP; lane 3: 25 ng rhTBP. Download figure Download PowerPoint Transcription of the yeast U6 gene reconstituted with yB″ activity, highly purified ypol III and recombinant yTFIIIB70 was completely dependent upon TBP (Figure 5B, lane 4) but could likewise be restored equally well by the addition of 25 ng recombinant TBP from yeast (Figure 5B, lane 3) or human (Figure 5B, lane 5). Experiments in which yeast and human TBP were titrated between 12.5 and 50 ng confirmed this result (data not shown). Mutant human TBP (R231K) but not wild-type hTBP can replace yeast TBP in the transcription of yeast tRNA genes in vitro Identical concentrations (25 ng) of the same recombinant TBP preparations from yeast and human, used above for transcription of human and yeast U6 genes (Figure 5A and B), were used for transcription of either the yeast Leu3 tRNA gene (data not shown) or the yeast SUP4 tRNA gene in a system which was entirely dependent on TBP (Figure 5C, lane 2). It was found that only yeast TBP showed full transcription activity but that human TBP resulted in very weak, if any transcription of both yeast tRNA genes examined (Figure 5C, compare lanes 1 and 3). It could be argued that this finding is the result of a sub-optimal concentration of human TBP in the transcription reaction. This is, however, not the case since reconstitution of SUP4 tRNA transcription by comparative titration of yeast and human TBP from 12.5 to 50 ng (Figure 6, compare lanes 2–4 and 5–7) confirmed the result of Figure 5C. Figure 6.Mutant human TBP-R231K is able to replace yeast TBP for SUP4 ytRNA and yU6 transcription in vitro. In lanes 1–10 the following protein fractions were incubated with 200 ng of pRS316 (SUP4 ytRNA gene): 100 ng ryTFIIIB70, 400 ng yTFIIIB″, 50 ng yTFIIIC and 50 ng ypolC (III) in all reactions. Lane 1 contains no TBP; lanes 2–4 contain 12.5, 25 and 50 ng recombinant yeast TBP; lanes 5–7 contain 12.5, 25 and 50 ng recombinant human wild-type TBP; lanes 8–10 contain 12.5, 25 and 50 ng recombinant mutant human TBP-R231K. Lane 11: labeled marker DNA [pBR322 DNA–MspI; sizes of fragments in nucleotides (nt) are appropriately indicated]; lanes 12–15: transcription of the yU6 gene. All reactions contained 100 ng ryTFIIIB70, 400 ng yTFIIIB″ and 50 ng ypol III. Lane 12 contains no TBP, lanes 13–15 contain 25 ng ryTBP, rhTBP and rhTBP-R231K respectively. Download figure Download PowerPoint It has previously been shown that human TBP, mutated at position 231 from Arg to Lys, is able to complement the defects of yeast TBP mutants in vivo (Cormack et al., 1994). Since our in vitro results pointed to a specific inability of human TBP to support tRNA transcription together with the heterologous yeast transcription factors, we analyzed the recombinant human mutant TBP-R231K for its ability to replace yeast TBP in pol III transcription. Indeed, we found that human TBP-R231K was able to replace yeast TBP for in vitro transcription of the yeast SUP4 tRNA gene (Figure 6, lanes 8–10), as well as the yeast U6 gene (Figure 6, lane 15). Discussion The B″ component of TFIIIB from S.cerevisiae can replace human TFIIIB for transcription of the human U6 gene For reconstituted in vitro transcription of the human U6 RNA gene, yeast and human TBP molecules are functionally interchangeable and hTFIIIB-α can be replaced by yB″. These data imply that yeast TBP and proteins in the yeast B″ fraction are able to productively interact with basal components of the evolutionary divergent human pol III transcriptional machinery. Interactions of yB″ with components of human TFIIIB are a prerequisite to establish a hybrid TFIIIB activity, which is required to correctly express the human U6 gene by RNA polymerase III. It is hence likely that essential domains of yeast B″ and hTFIIIB-α have been structurally conserved during the evolution of these highly diverged eukaryotes. In contrast to hTFIIIB-α, hTFIIIB-β cannot be functionally replaced by yeast TFIIIB or any of its components. Hence we did not find a functional conservation between yeast and human of such TFIIIB proteins, which are required for the transcription of classical pol III genes. We cannot exclude, however, that a human component, which is homologous to or fulfils the function of yB″ in the transcription of these pol III genes could also be present in the partially purified fractions used for reconstitution of tRNA transcription. Accordingly, it has been reported that B″ activity, sufficient for in vitro transcription of a yeast tRNA gene, can also be found in partially purified TFIIIC and pol III fractions (Kassavetis et al., 1991). Is hTFIIIB90 involved in transcription of the human U6 gene? Human TFIIIB90, which shows sequence homology to yeast TFIIIB70 (Wang and Roeder, 1995; Mital et al., 1996), is required for transcription of human pol III genes regulated