Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase.

We reported previously that cell-free transcription in the Archaea Methanococcus and Pyrococcus depends upon two archaeal transcription factors, archaeal transcription factor A (aTFA) and archaeal transcription factor B (aTFB). In the genome of Pyrococcus genes encoding putative homologues of eucaryal transcription factors TATA-binding protein (TBP) and TFIIB have been detected. Here, we report that Escherichia coli synthesized Pyrococcus homologues of TBP and TFIIB are able to replace endogenous aTFB and aTFA in cell-free transcription reactions. Antibodies raised against archaeal TBP and TFIIB bind to polypeptides of identical molecular mass in the aTFB and aTFA fraction. These data identify aTFB as archaeal TBP and aTFA as the archaeal homologue of TFIIB. At the Pyrococcus glutamate dehydrogenase (gdh) promoter these two bacterially produced transcription factors and endogenous RNA polymerase are sufficient to direct accurate and active initiation of transcription. DNase I protection experiments revealed Pyrococcus-TBP producing a characteristic footprint between position −20 and −34 centered around the TATA box of gdh promoter. Pyrococcus-TFIIB did not bind to the TATA box but bound cooperatively with Pyrococcus-TBP generating an extended DNase I footprinting pattern ranging from position −19 to −42. These data suggest that the Pyrococcus homologue of TFIIB associates with the TBP-promoter binary complex as its eucaryal counterpart, but in contrast to eucaryal TFIIB, it causes an extension of the protection to the region upstream of the TATA box.

Recent work established that cell-free transcription in Archaea is mediated by transcription factors (1)(2)(3). In the Euryarchaeon (4) Methanococcus two distinct archaeal transcription factors aTFA 1 and aTFB have been identified (1,5). Highly purified Methanococcus aTFB showed striking similarities to eucaryal TATA-binding proteins (TBP). It exists as a dimer in solution (5), can be replaced by yeast and human TBPs in cell-free transcription reactions (6), and binds in mobility gel shift assays (7) to DNA fragments harboring an archaeal (8,9) or eucaryal TATA box. Furthermore, the protein translation of a putative TBP homologue encoded in the genome of Thermococcus (10) was able to substitute for Methanococcus aTFB in cell-free transcription reactions and showed serological crossreaction with this polypeptide (11). Owing to its low stability purification of the aTFA activity thus far was not possible, but the incubation of aTFB or eucaryal TBPs in combination with aTFA results in template commitment (7,6), suggesting that it binds to and stabilizes the aTFB-promoter complex.
We have recently described a cell-free transcription system for the hyperthermophilic Archaeon Pyrococcus furiosus (12). In this system, specific transcription was as well dependent upon the presence of aTFB and aTFA activities. The discovery of two genes encoding putative homologues of eucaryal TBP and RNA polymerase II transcription factor B (TFIIB) in the genome of Pyrococcus (13)(14)(15) prompted us to investigate the function of these archaeal homologues of eucaryal transcription factors in promoter recognition and transcription and to determine their relationship to transcription factors purified from Pyrococcus cells. Our results indicate that aTFB and aTFA are identical with the Pyrococcus homologues of TBP and TFIIB. We report the DNase I protection patterns of the Pyrococcus TBP-DNA binary and TBP-TFIIB-DNA ternary complex.

EXPERIMENTAL PROCEDURES
Templates for Cell-free Transcription Reactions-The plasmid pLUW479 containing the promoter and a part of the coding region of the gdh gene of Pyrococcus furiosus was used in standard transcription reactions (12). DNA was purified by repeated centrifugation in CsCl density gradients as described previously (16).
Preparation of Transcription Factors and RNA Polymerase-The components required for cell-free transcription were purified as described previously (12). For further purification of RNA polymerase, the Superdex 200 fractions containing RNA polymerase activity were dialyzed against PS buffer (40 mM KPO 4 , pH 7.5, 1 mM EDTA, 0.1% Triton X-100) containing 1.5 M NaCl and were applied to a phenyl-Sepharose CL-4B column (0.5 ϫ 10 cm, Pharmacia Biotech Inc.) equilibrated with the same buffer. After washing the column with PS buffer containing 0.5 M NaCl and PS buffer without salt, bound RNA polymerase was eluted with PS buffer containing 10% ethylene glycol.
Expression and Purification of the Pyrococcus Homologue of TFIIB-A DNA fragment encoding the open reading frame of Pyrococcus woesei TFIIB gene was amplified by using the polymerase chain reaction with the following oligonucleotides: 5Ј-GGAATTCCATAT-GAATAAGCAAAAGGTTTGTC-3Ј and 5Ј-GTCATTCGAATTCATGC-TATAGGAACTTTAATC-3Ј. The amplified product was hydrolyzed with NdeI and EcoRI (restriction sites are underlined in the oligonucleotides). The hydrolyzed fragment was then ligated to pET17b (Novagen), which had been cleaved with NdeI and EcoRI. The resulting clone pTFIIBPW.17 was transformed into the Escherichia coli strain BL21(DE3) pLysS (17). The expression of the gene was induced by addition of isopropyl-1-thio-␤-D-galactopyranoside (final concentration: 1 mM) to a growing culture (A 600 ϭ 0.7) at 30°C. After induction for 4 h the cells were harvested by centrifugation. For purification a cell extract was prepared, heated for 15 min at 75°C, and subsequently * This work was supported by the European Community Biotechnology Program, a grant from the Deutsche Forschungsgemeinschaft, and by Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Expression and Purification of E. coli Produced TBP of P. woesei-The expression of the TBP was performed as for TFIIB with the following modifications. For amplification of the coding region the following oligonucleotides were used: 5Ј-GGAATTCCATATGGTGGATATGAG-CAAGG-3Ј and 5Ј-GGAATTCCTCAAAGCTCCTCCTCTTC-3Ј. The resulting clone pTBPPW.17 was transformed into the E. coli strain BL21(DE3), induction of gene expression was performed for 3 h, and the cell extract was heated for 15 min at 80°C. The recombinant TBP was purified from the supernatant by chromatography on Mono Q and Superdex 200 as described previously (11). The final preparation was about 90% pure.
Cell-free Transcription Reactions-The in vitro transcription reactions were performed as described previously (12). A standard reaction mixture (50 l) contained 1 g of linearized template DNA (pLUW479), 5 l of the phenyl-Sepharose fraction of the RNA polymerase, 5 l (0.43 unit) of the Superdex 200 fraction of aTFB, and 5 l (0.66 unit) of the heparin-Sepharose fraction of aTFA (12).
Preparation of Antibodies-500 g of recombinant TFIIB were electrophoresed in a preparative 12% denaturing polyacrylamide gel. The Coomassie Brilliant Blue-stained Pc-TFIIB was eluted from the gel and used for immunization of a rabbit. Immunization was performed by Eurogentec (Seraing, Belgium) following the standard immunization protocol. The polyclonal IgG fraction of the anti-TFIIB serum was purified by protein A-Sepharose chromatography (Pharmacia). For detection of Pyrococcus TBP in the aTFB fraction antibodies raised against Thermococcus TBP (11) were used.
Western Blot Analyses-Western blot analyses with antibodies directed against recombinant Thermococcus TBP and Pyrococcus TFIIB were performed as described previously (11).
DNase  4 Ac, 70 mM EDTA, 2 g of poly(dI-dC). The DNA fragments were purified by phenol treatment, precipitated with ethanol, and analyzed on a 6% sequencing gel (45 watts for 2.5 h).

RESULTS
To investigate the relationship of the factors encoded in the genome of Pyrococcus that are related to eucaryal TBP and TFIIB with Pyrococcus transcription factors aTFA and aTFB, the cloned Pyrococcus genes were overexpressed in E. coli, the polypeptides purified to near homogeneity, and their function analyzed in the Pyrococcus cell-free system. When a recombinant plasmid that is harboring the glutamate dehydrogenase gene of Pc. furiosus linearized with BamHI was used as template, a run-off transcript of 173 nucleotides was synthesized by the reconstituted Pyrococcus cell-free transcription system (Ref. 12; Fig. 1A, lane 7). This system consists of highly purified RNA polymerase (see "Experimental Procedures"), the Superdex fraction of aTFB and aTFA purified from the crude extract by a two-step procedure (12). Besides RNA polymerase both aTFA and aTFB activities are required for specific transcription (Fig. 1A, lanes 1-7). When a TFA was replaced by recombinant Pc-TBP, synthesis of a distinct RNA product was not observed (Fig. 1A, lane 8). However, the addition of Pc-TBP activated the archaeal system reconstituted without aTFB (Fig. 1A, lane 9). An increasing amount of Pc-TBP led to increased synthesis of the run-off transcript (Fig. 1A, lanes 9 and  10). This finding demonstrated that the TBP encoding gene detected in the genome of Pyrococcus encodes a transcription factor directing transcription from an archaeal promoter by Pyrococcus RNA polymerase. To investigate the function of the Pc-TFIIB homologue, both aTFA and aTFB were replaced with Pc-TFIIB. Pc-TFIIB was unable to substitute for aTFB (Fig.  1B, lane 1). However, a cell-free system reconstituted with Pc-TFIIB, aTFB, and RNA polymerase was able to direct specific transcription, and this Pc-TFIIB-dependent transcriptional activation was increased by adding increasing amounts of this recombinant polypeptide to cell-free transcription reactions (Fig. 1B, lanes 2 and 3). To address the question whether the aTFA and aTFB fractions harbor additional activities necessary for cell-free transcription, both archaeal factor fractions were replaced by the corresponding recombinant proteins. Pyrococcus RNA polymerase was able to synthesize an RNA product of correct size from the gdh promoter with high activity both in the presence of Pc-TBP and Pc-TFIIB (Fig. 1C, lane 4; compare with control lane 5). This finding demonstrates that Pc-TBP and Pc-TFIIB are able to replace the endogenous aTFA and aTFB fractions. Therefore, no additional activities contained in the aTFA and aTFB fraction are required for cell-free transcription of gdh promoter.
To investigate the relationship of aTFA and aTFB with Pc-TPB and Pc-TFIIB at the structural level, polyclonal antibodies raised against Thermococcus TBP (11) and Pc-TFIIB were probed with protein immunoblots of the three components of the Pyrococcus cell-free system. Recombinant Pc-TBP shows a molecular mass of 21.3 kDa. The antibody against Tc-TBP binds to a polypeptide of the same size in the aTFB fraction ( Fig. 2A, lane 1), but not in the aTFA or RNA polymerase fraction ( Fig. 2A, lanes 2 and 3). Recombinant Pc-TFIIB showed an apparent molecular mass of 34.1 kDa. Vice versa, a crossreacting polypeptide that comigrates during denaturing gel electrophoresis with Pc-TFIIB was found in the aTFA (Fig. 2B,  lanes 2 and 3) but not in the aTFB and RNA polymerase fraction (Fig. 2B, lanes 4 and 5). The aTFA fraction, however, contained additional cross-reacting polypeptides of lower molecular mass (Fig. 2B, lane 3), which are present as well in crude and more purified aTFA fractions (data not shown). The observation that both the aTFB and aTFA fractions contained polypeptides serologically related with Pc-TBP and Pc-TFIIB suggests that the factors activating the gdh promoter in the aTFA and aTFB fraction are identical with Pc-TFIIB and Pc-TBP.
To investigate the interaction of Pc-TBP and Pc-TFIIB with an archaeal promoter at the molecular level, binding of these factors to a 131-nucleotide end-labeled DNA fragment harboring the Pyrococcus gdh promoter was studied in DNase I protection assays. When 2.1 g of Pc-TBP were incubated with gdh promoter in DNA binding reactions, a clear DNase I footprint was detected. The footprint extended from position Ϫ20 to Ϫ34 and included the TATA box of this promoter located between position Ϫ22 and Ϫ30 (Fig. 3, compare control reactions in lanes 1-3 with lanes 4 -6). When Pc-TFIIB was incubated with this DNA fragment, specific binding to the TATA box was not observed, but a weak protection in the region located between position Ϫ4 and Ϫ6 was detected (Fig. 3, lanes 7-9). However, when Pc-TFIIB was added to binding reactions containing Pc-TBP, protection from DNase I digestion was significantly increased in the region of the TATA box and the 5Ј boundary of the footprint was extended from position Ϫ34 to Ϫ42 and the 3Ј boundary from position Ϫ20 to Ϫ19 (Fig. 3, lanes 10 -12). In addition, a hypersensitivity site located between position Ϫ5 and Ϫ8 was generated. In the presence of Pc-TFIIB, a smaller amount (0.7 g) of Pc-TBP was needed to protect the TATA box, and the protection in this DNA region was significantly increased (Fig. 3, lanes 10 -12) when compared with the protection pattern generated by Pc-TBP (Fig. 3, lanes 4 -6). These findings demonstrate that the archaeal TBP is the polypeptide interacting directly with the archaeal TATA box. The archaeal TFIIB homologue associates with the Pc-TBP-DNA complex and causes an extension of the protection seen with Pc-TBP. DISCUSSION Analysis of hybrid archaeal/archaeal and archaeal/eucaryal transcription systems provided evidence that aTFB is a highly conserved polypeptide analogous in function to eucaryal TBPs. aTFB is functionally interchangeable in the Methanococcus and Pyrococcus cell-free systems (12). 2 In the Methanoccoccus system aTFB can be replaced by the translation product of the putative Thermococcus TBP gene (11) and by yeast and human TBP (6). Pyrococcus aTFB can be replaced in the Pyrococcus system by recombinant Thermococcus TBP (12) and recombinant Pc-TBP (Fig. 1A). The sequence identity of the genes encoding archaeal TBPs with genes coding for eucaryal TBPs ranges between 30 and 35% (18). Similar as eucaryal TBPs all archaeal TBPs consist of a tandem repeat of two conserved domains, which are most likely the product of an ancient direct repeat (10). Methanococcus aTFB, Pc-TBP, and Sulfolobus-TBP were shown to bind to DNA fragments harboring an archaeal 2 C. Hethke and M. Thomm, unpublished data. TATA box in gel shift assays (7,13,18,19), and the TATA box of a Pyrococcus promoter was identified in this study as the binding site for Pc-TBP by DNase I protection studies (Fig. 3). All this evidence supports the conclusion that aTFB is an archaeal TBP analogous in function and homologous to eucaryal TBPs.
The nature of the second archaeal transcription factor, aTFA, was less clear. Archaea consist of two major phylogenetic lineages, the Euryarchaeota comprising methanogens and Pyrococcus and the Crenarchaeota (4). In the Crenarchaeon Sulfolobus a second transcription factor activity has not yet been identified, although a gene with significant sequence similarity to eucaryal TFIIB exists in the genome of this organism (20). Both Methanococcus and Pyrococcus aTFA separate at early steps of purification from RNA polymerase (1,12) and are rapidly inactivated when purified by more than one or two chromatographic purification steps. 3 Pyrococcus and Methanococcus aTFA are species specific. 2 In this study, we show that Pyrococcus aTFA activity can be replaced by a single polypeptide displaying significant amino acid similarity with RNA polymerase II transcription factor TFIIB (Fig. 1, B and C). The gene encoding this Pc-TFIIB factor shows an identity of 32-36% with TFIIB from various eucaryal sources. In addition, this sequence displays unique structural motifs characteristic of eucaryal TFIIB such as an imperfect amino acid repeat in the N and C terminus of the molecule and a zinc II finger motif located close to the N terminus (14,15). This Pyrococcus homologue of TFIIB produced in E. coli showed serological crossreaction with a polypeptide of identical molecular mass in the aTFA fraction (Fig. 2B). The origin of the additional crossreacting polypeptides of lower molecular mass in this fraction (Fig. 2B, lane 3) is unclear, but we have demonstrated that a single polypeptide is able to replace the aTFA activity in cellfree transcription reactions (Fig. 1B). Taken together these data provide strong evidence that the second archaeal transcription factor, aTFA, can be defined as archaeal homologue of eucaryal TFIIB. At the gdh promoter, these two factors and purified RNA polymerase are sufficient to direct specific transcription. It seems unlikely that the enzyme contained additional transcription factors, as only polypeptides identified as general components of archaeal RNA polymerases (21) were found in silver-stained denaturing polyacrylamide gels of purified Pyrococcus RNA polymerase fractions (data not shown).
Pc-TBP and Pc-TFIIB show also strong similarity to eucaryal TBP and TFIIB in their mode of interaction with a TATA box containing promoter. Human TBP protects a 20-nucleotide region centered at the TATA box from DNase I digestion (22), archaeal TBP 15 nucleotides as well centered at the TATA box (Fig. 3). Similar to eucaryal TFIIB, the archaeal homologue of this factor shows no intrinsic ability to bind to the TATA box, although a weak protection in the region downstream of the TATA box was detected (Fig. 3, lanes 7-9). But, similar to TFIIB, it can associate with a binary complex of TBP and TATA box, resulting in the formation of a DNA-TBP-TFIIB ternary complex as has been shown by gel shift analyses (13) and footprinting studies (Fig. 3). Formation of this ternary complex at the archaeal promoter is characterized by an increase in the nucleotide region protected from DNase cleavage. Association of eucaryal TFIIB with the TBP-DNA complex leads also to an increase of DNA protected from DNase I or chemical cleavage (23,24). Inclusion of archaeal TFIIB into the archaeal TBPpromoter complex clearly induces an additional protection of 8 nucleotides at the 5Ј end and of only one nucleotide at the 3Ј end of the footprint (see Fig. 3, to the right). Although it is unclear whether this additional region protected in the ternary complex is bound by Pc-TFIIB, Pc-TBP, or both, a clear difference to the eucaryal system becomes apparent. DNase I footprinting experiments demonstrated that eucaryal TFIIB in the ternary complex causes an extension of the footprint to the DNA region downstream of the TATA element, but similar to Pc-TFIIB, eucaryal TFIIB stabilizes the TBP-DNA binary complex (Ref. 25,reviewed in Ref. 26). In spite of this difference the first steps of promoter activation and the central core of transcriptional machinery seem to be very similar in Archaea and Eucarya. In vitro studies with negatively supercoiled templates demonstrated that TBP and TFIIB represent the minimal set of factors directing RNA polymerase to the promoter (27). These components are conserved among Archaea and Eucarya, and the assembly of these components at the promoter occurs in Archaea in the same sequence as in Eucarya. An archaeal TBP recognizes the TATA motif, an archaeal TFIIB homologue binds to this complex, and this association of TFIIB with the binary complex results in stabilization of the TBP-DNA interaction. Both the increased resistance of the Pc-TBP-Pc-TFIIB ternary complex to DNase I cleavage (compare Fig. 3, lanes 4 -6 with 10 -12) and the finding that less Pc-TBP than in the binary complex is required to protect the TATA box in the ternary complex demonstrate that Pc-TFIIB stabilizes binding of TBP to the TATA box. These similarities in the DNA interaction of homologous components of basal transcriptional ma-  [13][14][15][16] show sequencing reactions of the gdh promoter fragment. The same probe was used as for footprinting experiments, but the fragment used for cycle sequencing reactions was internally labeled with 33 P at adenine residues. Those residues that are protected from DNase I digestion in DNA-Pc-TBP binary and in DNA-Pc-TBP-Pc-TFIIB ternary complexes are bracketed to the right of the figure. The arrow indicates direction of transcription of the gdh template. The positions and sequences of protected DNA segments relative to the transcription start site (ϩ1) are indicated. chinery suggest that the archaeal and eucaryal transcriptional machineries are of the same evolutionary origin. The archaeal transcription apparatus seems to represent the conserved version of the primordial eucaryal transcription apparatus, and analysis of transcription in Archaea therefore is likely to shed new light on the molecular evolution of transcriptional machineries in eucaryal cells. To indicate the homology of archaeal and eucaryal transcription factors, we suggest to designate aTFB as archaeal TBP (aTBP) and aTFA as transcription factor B (TFB).