The TATA-binding protein (TBP) is a principal component of the general factor TFIID and is required for specific transcription by RNA polymerase II. We have shown that TBP is also a general factor for RNA poly-merase III.

RNA polymerase III is responsible for the synthesis of a variety of small RNA molecules, including tRNA and 5 S rRNA. This transcription is coordinately regulated in response to a number of environmental stimuli, such as cell growth (Mauck and Green, 1974), differentiation (White et al., 1989) and transformation (White et al., 1990). In each of these cases, coordinate regulation is achieved by modulation of the activity of one or more of the general class III transcription factors (Carey and Singh, 1988; Hoeffler et al., 1988; Tower and Sollner-Webb, 1988; White et al., 1989; White et al., 1990). (Class III genes are those transcribed by RNA polymerase III, class II and I genes are those transcribed by RNA polymerases II and I, respectively). In order to achieve a clear understanding of such regulation, it is important to obtain a detailed knowledge of how many factors are involved in the basal transcription machinery.

The first estimate of the complexity of the class III transcription apparatus was gained as a result of fractionating extracts of human KB cells (Segall et al., 1980). The initial stage in this fractionation scheme involved chromatography on phosphocellulose and is schematised in Fig. 1. Two step fractions, PC-B and PC-C, were found to be necessary and sufficient to reconstitute transcription of a tRNA gene and an adenovirus VA gene in the presence of purified RNA polymerase III. Synthesis of 5 S rRNA also required these two fractions, as well as an additional fraction, PC-A. On this basis it was concluded that the PC-A fraction contains a gene-specific factor, which was named TFIIIA, whereas the PC-B and PC-C fractions each contain a general factor, which were named TFIIIB and TFIIIC, respectively (Segall et al., 1980). Since these experiments employed crude step fractions, the possibility of additional class III factors within the various fractions had not been excluded. Indeed two further factors have since been identified in silkworms (Ottonello et al., 1987; Young et al., 1991). However, in no other system were any additional general class III initiation factors identified for over a decade, despite many fractionation studies.

Fig. 1.

Simplified version of fractionation scheme used by Segall et al. (1980) to separate class III transcription factors from an S100 extract of cultured human KB cells. The KCI concentrations at which the fractions eluted are indicated.

Fig. 1.

Simplified version of fractionation scheme used by Segall et al. (1980) to separate class III transcription factors from an S100 extract of cultured human KB cells. The KCI concentrations at which the fractions eluted are indicated.

TBP is required for TATA-dependent transcription of U6 snRNA genes by RNA polymerase III

Extensive purification of a yeast TFIIIB preparation resolved two components, neither of which was sufficient on its own to support transcription of a U6 snRNA template by RNA polymerase III (Margottin et al., 1991). Whereas one of these components possessed TFIIIB activity, the other resembled the TATA-binding protein (TBP) in terms of its size, TATA-binding properties and ability to direct transcription of the adenovirus major late gene by RNA polymerase II in a TBP-dependent system. Furthermore, cloned TBP could substitute for this component in reconstituting RNA polymerase III transcription of the U6 snRNA gene. The involvement of TBP in U6 gene transcription is consistent with the presence of a TATA box ∼30 bp upstream of the transcription start site. In the case of human and Xenopus U6 genes, this sequence has been shown to be important for polymerase III transcription (Carbon et al., 1987; Mattaj et al., 1988; Lobo and Hernandez, 1989; Simmen and Mattaj, 1990; Simmen et al., 1991). Indeed, the TATA box elements of several class II genes can functionally substitute for the U6 TATA sequence in directing transcription by RNA polymerase III (Lobo et al., 1991). As was the case for the yeast gene, addition of purified or cloned TBP was found to activate polymerase III transcription of the human U6 gene in a variety of reconstituted systems (Lobo et al., 1991; Simmen et al., 1991; Waldschmidt et al., 1991).

TBP: an unlikely candidate for a general class III factor

The presence of an essential TATA box in the promoters of U6 genes had suggested the possibility of a role for TBP, although a distinct TATA-binding factor for RNA polymerase HI had, for a time, been considered likely (Carbon et al., 1987; Mattaj et al., 1988; Murphy et al., 1989; Simmen and Mattaj, 1990). Functional TATA boxes are also found in several other polymerase III promoters, such as those of the human 7SK gene (Murphy et al., 1987) and the Epstein-Barr virus EBER2 gene (Howe and Shu, 1989). In contrast, most class III genes have no TATA homology and generally have much less reliance on specific sequences upstream of the transcription start site (reviewed by Murphy et al., 1989 and Gabrielsen and Sentenac, 1991). The absence of a TATA box in most class III genes made TBP seem an unlikely candidate for a general class III transcription factor. However, transcription of a TATA-less template by RNA polymerase II was shown, nevertheless, to require the presence of TBP (Pugh and Tjian, 1991). Since this was the case for RNA polymerase II, it could clearly also be so for RNA polymerase III.

A second argument against TBP as a general class III factor was the fact that TFIID, of which TBP is a principal component, elutes from phosphocellulose in the PC-D fraction, which is not required for polymerase III transcription (Fig. 1; Matsui et al., 1980). However, western blotting with affinity-purified, anti-TBP antibodies has shown that TBP is, in fact, also present in significant amounts in both PC-B and PC-C, the fractions which are generally required by class III genes (R. J. White and S. P. Jackson, unpublished results). Therefore, a subpopulation of TBP molecules cofractionates with the established polymerase III general factors. So TBP could no longer be excluded, a priori, as a general class III factor.

A TATA-binding factor is required for transcription of all class III genes tested

We tested for an involvement of TBP in general polymerase III transcription using TATA-containing oligonucleotides.

Preincubation of a HeLa nuclear extract with an oligonucleotide containing the sequence of the TATA box region of the adenovirus major late (ML) promoter inhibits transcription of a subsequently added human U6 snRNA gene; this effect is TATA-specific, since point-mutating the TATA box whilst leaving the remainder of the oligonu-cleotide unchanged abolishes the inhibition (Fig. 2, lanes 9 and 10). The sensitivity of U6 transcription to competing TATA sequences reflects its known requirement for TBP (Lobo et al., 1991; Margottin et al., 1991; Simmen et al., 1991; Waldschmidt et al., 1991). Transcription of the Epstein-Barr virus EBER2 gene shows the same response (Fig. 2, lanes 7 and 8). A TATA box positioned 28 bp upstream of the start site of this gene has been shown to make a major contribution to EBER2 expression in vivo (Howe and Shu, 1989). Transcription of a member of the B2 middle-repetitive gene family is also specifically inhibited by the TATA sequence (Fig. 2, lanes 5 and 6). This gene has an internal promoter, but also has a TATA homology 15 bp upstream of the transcription start site (White,1990). However, transcription of a human Alu gene and a Caenorhabditis elegans tRNAPro gene, neither of which have TATA boxes (Ciliberto et al., 1982; C. Sharpe, personal communication), is also specifically inhibited by the ML TATA sequence (Fig. 2, lanes 1 – 4).

Fig. 2.

Effect of oligonucleotide competitors upon RNA polymerase III transcription. tRNAPro (lanes 1 – 2), Alu (lanes 3 – 4), B2 (lanes 5 – 6), EBER2 (lanes 7 – 8) and U6 (lanes 9 – 10) genes were transcribed using HeLa nuclear extract that had been preincubated for 15 min at 30°C with 400 ng of ML TATA (lanes 1,3, 5, 7 and 9) or ML TATA mutant (lanes 2, 4, 6, 8 and 10) oligonucleotides. The additional band in all lanes, that is marked by an asterisk and does not respond to competition, is due to end-labelling of endogenous small RNAs (G. Kunkel, personal communication). The ML TATA and ML TATA mutant oligonucleotides are as previously (White et al., 1992). The tRNAPro template is plasmid Mcetl (Ciliberto et al., 1982). The Alu template is pRH5.7, a 5.7 kb fragment of human genomic DNA from the apoAl-CIII locus inserted into the Smal site of pAT153. The B2 template is pTB14 (White et al., 1989). The EBER2 template is E2,-160 (Howe and Shu, 1989). The U6 template is pU6/Hae/RA.2 (Lobo et al., 1991). Extracts were prepared as previously (White et al., 1992). Transcription reactions were performed and processed as previously (White et al., 1992).

Fig. 2.

Effect of oligonucleotide competitors upon RNA polymerase III transcription. tRNAPro (lanes 1 – 2), Alu (lanes 3 – 4), B2 (lanes 5 – 6), EBER2 (lanes 7 – 8) and U6 (lanes 9 – 10) genes were transcribed using HeLa nuclear extract that had been preincubated for 15 min at 30°C with 400 ng of ML TATA (lanes 1,3, 5, 7 and 9) or ML TATA mutant (lanes 2, 4, 6, 8 and 10) oligonucleotides. The additional band in all lanes, that is marked by an asterisk and does not respond to competition, is due to end-labelling of endogenous small RNAs (G. Kunkel, personal communication). The ML TATA and ML TATA mutant oligonucleotides are as previously (White et al., 1992). The tRNAPro template is plasmid Mcetl (Ciliberto et al., 1982). The Alu template is pRH5.7, a 5.7 kb fragment of human genomic DNA from the apoAl-CIII locus inserted into the Smal site of pAT153. The B2 template is pTB14 (White et al., 1989). The EBER2 template is E2,-160 (Howe and Shu, 1989). The U6 template is pU6/Hae/RA.2 (Lobo et al., 1991). Extracts were prepared as previously (White et al., 1992). Transcription reactions were performed and processed as previously (White et al., 1992).

The fact that a TATA sequence specifically competes for transcription of these genes indicates that a TATA-binding factor is required for their expression. This is clearly a general effect, since it is shown by five different class III genes, with a range of promoter structures (Fig. 2). Strikingly, two of these genes have no TATA boxes and yet still require a TATA-binding factor for transcription. We have obtained the same response with six other TATA-less polymerase III templates, including the well-characterised adenovirus VAi and Xenopus borealis somatic 5 S rRNA genes (White et al., 1992). In fact, transcription of all of the class III genes that we have tested is specifically inhibited by competition with TATA sequences. Therefore, there is a general polymerase III transcription factor that recognizes TATA boxes.

A general class III transcription factor has DNA-binding specificity and antigenic epitopes in common with TBP

It was important to exclude the possibility that the ML TATA sequence represents a cryptic binding site for one of the established class III general factors, TFIIIB and TFIIIC. TFIIIC has well-characterised DNA-binding properties, whereas TFIIIB by itself shows no sequence-specific interaction with DNA (reviewed by Gabrielsen and Sen-tenac, 1991). We therefore tested whether TFIIIC could recognize TATA boxes in a gel retardation assay and found that the ML TATA oligonucleotide was unable to compete for TFIIIC binding (R. J. White and S. P. Jackson, unpublished results). Therefore, the factor that recognizes TATA boxes and is required for transcription of all polymerase III templates is unlikely to be one of the established class III general factors.

Cloned human TBP binds to the ML TATA box and a point-mutation that abolishes the inhibitory effect of this sequence on polymerase III transcription (TATAAAA→ TAGAGAA) also abolishes TBP binding (Hoffmann et al., 1990). This correlation was extended further by the observation that functional TATA boxes from two other well-characterised polymerase II promoters also inhibit transcription of all polymerase III templates tested (White et al., 1992). Thus, polymerase III transcription is inhibited by the CATATAA sequence that serves as a TATA box in the mouse β-globin promoter and by the CATAAAA sequence that serves as a TATA box in the human p-globin promoter. Point-mutation of either of these sequences (CATATAA-ÆATAGCA and CATAAAA→CATCGCC) again abolishes the ability to inhibit transcription of class III genes (White et al., 1992). Therefore, the TATA-binding general factor required for transcription by RNA polymerase III specifically recognizes three different TATA boxes that serve as functional targets for TBP.

We also tested the effect on polymerase III transcription of physically removing TBP by immunodepletion. An extract that had been immunodepleted using affinity-purified antisera against cloned TBP was found to be severely impaired relative to a mock-immunodepleted control extract, in its ability to transcribe all polymerase III templates tested (R. J. White, B. F. Pugh and S. P. Jackson, unpublished results). These templates included the Xenopus borealis somatic 5 S rRNA gene, the adenovirus VAi gene and a human tRNALeu gene, all of which lack TATA sequences.

Cloned TBP restores RNA polymerase III transcription to extracts that have been preincubated with TATA competitors

The general class III factor that recognizes TATA boxes has similar or identical DNA-binding specificity to TBP and is recognized by affinity-purified antibodies against TBP. These observations strongly implicate TBP as this general factor. However, the alternative of a distinct class III TATA-binding factor with epitopes and DNA-binding specificity in common with TBP is not excluded by this data. It was therefore necessary to test whether TBP itself could function as a general factor for RNA polymerase III. TBP had already been shown to activate TATA-dependent transcription of the U6 snRNA gene (Lobo et al., 1991; Margottin et al., 1991; Simmen et al., 1991; Waldschmidt et al., 1991). However, none of the classical class III genes, without TATA boxes, such as VA, tRNA and 5 S rRNA, had been shown to respond to TBP.

Cloned human TBP (Peterson et al., 1990), expressed in bacteria and purified to >95% homogeneity, restores transcription of TATA-less polymerase III templates in extracts preincubated with TATA-containing oligonucleotides (White et al., 1992). This is shown in Fig. 3 for the VA1 gene and a B2 gene. The inclusion of 2 (μg/ml α-amanitin in this experiment excluded the possibility that TBP-induced transcription of these class III genes was by RNA polymerase II, since this concentration of α-amanitin selectively inhibits the polymerase II enzyme but not RNA polymerase III. Whereas cloned TBP can activate polymerase III transcription following preincubation of extracts with ML or β-globin TATA sequences, extracts preincubated with a TFIIIC binding site do not respond (White et al., 1992). This demonstrates the specificity of the effect and also confirms that TFIIIC is not the target for TATA box competitors.

Fig. 3.

Cloned TBP restores RNA polymerase III transcription to extracts depleted of TATA-binding proteins by preincubation with TATA oligonucleotides. VA1 (lanes 1 – 3) and B2 (lanes 4 – 6) genes were transcribed in the presence of 2 μ g/ml a-amanitin using HeLa extract preincubated for 15 min at 30°C with 200 ng of the ML TATA mutant (lanes 1 and 4) or the ML TATA (lanes 2, 3, 5 and 6) oligonucleotides. In lanes 3 and 6, template was preincubated separately with 8 ng of >95% pure cloned human TBP for 15 min at 30°C before being added to the depleted extract. The VA1 template was pBRVAj (White et al., 1989). Purified bacterially expressed cloned human TBP was a generous gift from B. F. Pugh and R. Tjian (Peterson et al., 1990).

Fig. 3.

Cloned TBP restores RNA polymerase III transcription to extracts depleted of TATA-binding proteins by preincubation with TATA oligonucleotides. VA1 (lanes 1 – 3) and B2 (lanes 4 – 6) genes were transcribed in the presence of 2 μ g/ml a-amanitin using HeLa extract preincubated for 15 min at 30°C with 200 ng of the ML TATA mutant (lanes 1 and 4) or the ML TATA (lanes 2, 3, 5 and 6) oligonucleotides. In lanes 3 and 6, template was preincubated separately with 8 ng of >95% pure cloned human TBP for 15 min at 30°C before being added to the depleted extract. The VA1 template was pBRVAj (White et al., 1989). Purified bacterially expressed cloned human TBP was a generous gift from B. F. Pugh and R. Tjian (Peterson et al., 1990).

The ability of cloned TBP to activate transcription of TATA-less polymerase III templates in extracts that have been depleted of endogenous TATA-binding factor(s) is strong evidence in support of the hypothesis that TBP is a general class III transcription factor. However, if an alternative TATA-binding factor were employed by RNA polymerase III, then addition of exogenous cloned TBP might simply serve to displace this factor from the TATA oligonu-cleotides, thereby allowing it to mediate transcription of class III genes. To exclude this possibility, we tested the effect of cloned TBP on polymerase III transcription in extracts in which endogenous TBP had been inactivated by heat treatment.

Cloned TBP restores RNA polymerase III transcription to heat-treated extracts

Mild heat treatment (47°C for 15 minutes) has been shown to inactivate TBP whilst leaving the other components of the class II general transcription apparatus unaffected (Nakajima et al., 1988). Such treatment abolishes transcription of all class III templates tested, consistent with a general requirement for TBP in polymerase III transcription (Fig. 4; Simmen et al., 1991; White et al., 1992; R. J. White and S. P. Jackson, unpublished observations). As well as TBP, a component of the PC-C fraction, presumably TFIIIC, is also inactivated in this way (Simmen et al.,1991)Addition of PC-C to the heat-treated extract elicits only a very slight restoration of transcription, but if cloned TBP is also included, polymerase III transcription is dramatically stimulated. This is shown in Fig. 4 for a B2 gene. It is also the case for U6 (Simmen et al., 1991), VAi and tRNA genes (White et al., 1992). Therefore, TBP can directly activate transcription of both TATA-containing and TATA-less templates. Indeed, under appropriate conditions, the PC-C fraction and cloned TBP are sufficient to restore fully levels of transcription in a heat-treated extract to the same level as those occurring in an unheated extract (Simmen et al., 1991: White et al., 1992). Heat treating the cloned TBP abolishes its ability to activate transcription of class III templates (White et al., 1992). The transcription reconstituted in a heat-inactivated extract using PC-C and TBP is abolished by 100 μ g/ml but not by 2 Ltg/ml α-amanitin, which is the α-amanitin sensitivity diagnostic of mammalian RNA polymerase III (White et al., 1992). Therefore, TBP can direct polymerase III transcription of a variety of class III genes.

Fig. 4.

Cloned TBP restores RNA polymerase III transcription to heat-inactivated extracts. A B2 gene was transcribed in the presence of 2 μ g/ml α-amanitin using either untreated (lane 1) or heat treated (lanes 2 – 4) HeLa extract in the presence (lanes 3 – 4) or absence (lanes 1 – 2) of 4 μ l of a PC-C fraction. Lane 4 contained 6.7 ng of cloned TBP. PC-C phosphocellulose column fraction was prepared according to the method of Segall et al. (1980). Heat treatment was for 15 min at 47°C according to Nakajima et al. (1988).

Fig. 4.

Cloned TBP restores RNA polymerase III transcription to heat-inactivated extracts. A B2 gene was transcribed in the presence of 2 μ g/ml α-amanitin using either untreated (lane 1) or heat treated (lanes 2 – 4) HeLa extract in the presence (lanes 3 – 4) or absence (lanes 1 – 2) of 4 μ l of a PC-C fraction. Lane 4 contained 6.7 ng of cloned TBP. PC-C phosphocellulose column fraction was prepared according to the method of Segall et al. (1980). Heat treatment was for 15 min at 47°C according to Nakajima et al. (1988).

TBP functions as a general class III transcription factor in vivo

Transcription of a broad range of class III genes therefore requires a factor with the same TATA-binding specificity as TBP, the same heat-sensitivity as TBP, with antigenic epitopes in common with TBP, and which can be functionally substituted for by cloned TBP. This strongly suggests that TBP is a general factor for RNA polymerase III. However, the above experiments were all performed in vitro. It therefore remained to confirm that TBP plays the same role in vivo. This has been achieved using yeast genetics.

Expression of U6 snRNA, tRNATrP and tRNAIle is rapidly inhibited following transfer of strains bearing temperature-sensitive mutations in TBP to the non-permissive temperature (Cormack and Struhl, 1992). A mutation in the gene encoding the largest subunit of RNA polymerase II does not produce a similar effect, suggesting that the response of U6 and tRNA transcription to TBP mutations is not an indirect consequence of a defect in polymerase II transcription. The rapid response of U6 and tRNA synthesis to inactivation of TBP is also consistent with a direct involvement of TBP in the expression of these genes (Cormack and Struhl, 1992). Furthermore, cell-free extracts made from yeast strains bearing mutated TBP are unable to transcribe 5 S rRNA or tRNALeu3 genes (Schultz et al.,1992). Addition of cloned TBP restores 5 S rRNA and tRNA transcription by these extracts, thereby proving that the TBP mutations are not acting indirectly (Schultz et al., 1992). These experiments conclusively demonstrate that TBP is a general transcription factor for RNA polymerase III in vivo.

TBP is a general transcription factor for RNA polymerase III. We have tested eleven different class III genes, including representatives of all the types of promoter organisation known to occur in polymerase III templates (reviewed by Gabrielsen and Sentenac, 1991), and in all cases we have observed a requirement for TBP. Furthermore, each of the yeast genes tested has also shown the same TBP-dependence (Cormack and Struhl, 1992; Schultz et al., 1992). Therefore, the TBP requirement for RNA polymerase III transcription is a general one.

Two general class III initiation factors, TFIIIB and TFIIIC, were defined in 1980 (Segall et al., 1980). Since that time, two new factors, TFIIID and TFIIIR, have been discovered in silkworms (Ottonello et al., 1987; Young et al., 1991). However, it remains to be determined whether these factors are ubiquitous or silkworm-specific. Furthermore, since TFIIID and TFIIIR were only tested on two templates (5 S rRNA and tRNAAla genes), it is not yet clear that these are truly general factors. Indeed, TFIIIC can no longer be rigorously included in the general category, since it is not required for transcription of U6 genes (Moenne et al., 1990; Lobo et al., 1991; Margottin et al., 1991; Wald-schmidt et al., 1991). At present, TFIIIB and TBP are the only class III initiation factors that can be considered to be truly general.

It has been reported that little or no free TBP exists in HeLa cell extracts (Timmers and Sharp, 1991). Instead, human TBP is found in large, multi-component complexes such as TFIID, which contain additional polypeptides that are tightly bound to TBP, and are consequently referred to as TBP-associated factors or TAFs (Pugh and Tjian, 1991; Tañese et al., 1991). It therefore seems likely that the TBP population that is active in RNA polymerase III transcription also exists in association with other proteins. The fact that a population of TBP molecules copurifies with TFIIIB activity in yeast (Margottin et al., 1991) suggests that TBP and TFIIIB may interact in solution, which further implies that they may interact in a functional transcription complex. It also seems likely that there are class Ill-specific TAFs associated with the TBP subpopulation that mediates RNA polymerase III transcription. If so, then these represent additional components of the class III transcription machinery that are awaiting discovery.

Since many class III promoters do not have TATA boxes, it is not clear how they recruit TBP. TBP is capable of specifically recognizing sequences that are quite different from a consensus TATA element (Singer et al., 1990). It is therefore possible that TBP binds directly to at least some TATA-less class III genes. However, since the only sequences that are obviously conserved between tRNA, VA and B2 genes are the A- and B-block internal promoter elements that are bound by TFIIIC, TBP would presumably have to recognise different sequences in different genes, were direct binding to be the rule. It seems far more probable that TBP is recruited to such genes by protein-protein interactions. Template-commitment experiments support this conclusion (R. J. White and S. P. Jackson, unpublished observations). This is in keeping with results for a TATA-less RNA polymerase II template, where TBP recruitment is achieved by interaction with a protein component of the TFIID complex, referred to as a ‘tethering’ factor (Pugh and Tjian, 1991).

TBP is also required for specific transcription by RNA polymerase I (Comai et al., 1992; Cormack and Struhl, 1992; Schultz et al., 1992). In this case the essential class I factor SL1 is composed of TBP and three TAFs (Cornai et al., 1992). Polymerase I templates do not have TATA homologies, but murine SL1 can bind directly to the mouse rRNA gene promoter (Bell et al., 1990). Which component of SL1 is responsible for this DNA binding remains to be determined. In contrast, human SL1 cannot bind independently to the human rRNA gene promoter, but needs to be tethered via protein-protein interactions with an upstream-binding factor termed UBF (Bell et al., 1988; Bell et al., 1990). Therefore, the human class I system provides a good example of TBP recruitment to a TATA-less promoter by protein-protein interactions.

It will be interesting to determine whether the different classes of transcription machinery utilise the same or different regions of the TBP polypeptide. The C-terminal 180 amino acids of TBP are at least 80% identical in all organisms from which it has been cloned; these include human, yeast (Saccharomyces cerevisiae and Schizosaccharomyces pombe), the fruit fly Drosophila melanogaster and the plant Arabidopsis thaliani (see Greenblatt, 1991, for a review). This conserved region has been shown to be sufficient to allow basal transcription in vitro from a TATA-containing promoter by RNA polymerase II (Hoey et al., 1990; Horikoshi et al., 1990; Peterson et al., 1990). We have found that it is also sufficient for RNA polymerase III transcription of a TATA-less tRNA gene (R. J. White, B. F. Pugh and S. P. Jackson, unpublished observations). Three different point-mutations within this region abolish transcription by each of the nuclear RNA polymerases in vivo, suggesting that much of the protein is important for all classes of transcription (Cormack and Struhl, 1992; Schultz et al., 1992). However, two other point-mutations produce class-specific effects: one abolishes transcription by RNA polymerases II and III but allows transcription by RNA polymerase I; another, which inactivates TATA binding, still functions with TATA-less class I and III genes but not with a TATA-containing class II promoter (Schultz et al., 1992). Therefore, components of the different classes of transcription machinery may contact overlapping but distinct regions of TBP.

For over a decade the TATA-binding protein was considered to be specifically required for TATA-dependent transcription by RNA polymerase II. Recently, its known sphere of influence has rapidly expanded to include TATA-dependent transcription by RNA polymerase III (Margottin et al., 1991), TATA-independent transcription by RNA polymerase II (Pugh and Tjian, 1991), TATA-independent transcription by RNA polymerase III (White et al., 1992) and, finally, transcription by RNA polymerase I (Cornai et al., 1992). These dramatic discoveries (reviewed by White and Jackson, 1992) have established TBP as the central component of nuclear transcription. It is unique in being a general factor required for transcription by RNA poly-merases I, II and III. The fact that it has maintained this position from yeast to man suggests that it will prove to be the case in all eukaryotes. If so, then transcription of all nuclear genes is likely to require this small, remarkable polypeptide.

We thank Frank Pugh and Robert Tjian for purified TBP, Colin Sharpe for pRH5.7, Nouria Hernandez for pU6/Hae/RA.2 and Gregory Howe for E2,-160. This work was funded by the Cancer Research Campaign and the Medical Research Council of Great Britain. R. J. W. and S. P. J. are members of the Zoology Department of Cambridge University. S. P. J. is also supported by a Leukaemia Society of America Senior Fellowship.

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