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 cellfree 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 (1-3). 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 (13-15) prompted u...

TATA boxes are common structural features of eucaryal class II and archaeal promoters. In addition, a gene encoding a polypeptide with sequence similarity to eucaryal TATA-binding protein (TBP) has recently been detected in Archaea, but its relationship to the archaeal transcription factors A (aTFA) and B (aTFB) was unclear. Here, we demonstrate that yeast and human TBP can substitute for aTFB in a Methanococcus-derived archaeal cell-free transcription system. Template-commitment studies show that eucaryal TBP is stably sequestered at the archaeal promoter and that this interaction is further stabilized in combination with aTFA. Binding studies revealed that recognition of an archaeal promoter by TBP involves specific binding to the TATA box. These findings demonstrate a common function of TBP and aTFB and imply a common evolutionary origin of eucaryal and archaeal transcriptional machinery.Comparative analyses of 16S rRNA sequences revealed the existence of two phylogenetically distinct groups of prokaryotes (1, 2), now called Archaea and Bacteria (3). Woese's concept of two prokaryotic domains differing from each other as much as from Eucarya (eukaryotes) was confirmed by investigating the biochemistry of these organisms (4) and by analyzing the sequences of molecules, such as translation elongation factors (5) and RNA polymerases (6). Recent studies based on rooted phylogenetic trees suggest that Archaea and Eucarya are specifically related (7,8).Several striking similarities between Archaea and Eucarya have been detected on the level of transcriptional machinery. First, Archaea have a single RNA polymerase resembling the RNA polymerases II and III of Eucarya (6,9). Second, archaeal promoters and most eucaryal RNA polymerase II promoters share a TATA box at position -25 as the major control region directing the initiation of transcription (10, 11). Interaction of TATA-binding protein (TBP) with the TATA box is the first step in the activation of RNA polymerase II promoters (12), and TATA-box-dependent initiation of transcription in Archaea has also been shown to require transcription factors (13)(14)(15) (yTBP) and human (hTBP) were synthesized in, and purified from, Escherichia coli (24). Yeast transcription factor IIA (TFIIA) was purified as described (25).Templates. All pIC31 derivatives (Fig. 1) were constructed, and their DNA sequences were verified, as published previously (10). pIC31/2 harbors the tRNAVal gene of Methanococcus vannielii; pIC31/11 is equivalent to pIC31/2, except that the first six bases of the promoter are deleted; pIC31/44 differs from the pIC31/2 by a T --G transversion at position 5 of the TATA box; pIC31/61 is equivalent to pIC31/2, except that internal control regions are replaced by a random DNA sequence derived from E. coli; and pIC31/64 is also derived from pIC31/2, but a DNA segment of 28 bp derived from E. coli was inserted at position +5. pNH1 harbors the dinitrogenase reductase gene of M. thermolithotrophicus (26, 27), and plasmid pKS304A16 contains the gene en...