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...

Our understanding of the mechanism of RNA biosynthesis in archaebacteria is limited, due in part to the inability of purified RNA polymerases to transcribe purified genes accurately in vitro. In the present study, we show that cell extracts of Methanococcus vannielii and Methanococcus thermolithotrophicus purified by gradient centrifugation synthesize a distinct transcript from templates harboring a cloned homologous tRNA(Val) and tRNA(Arg) gene. The in vitro transcripts initiate with GTP at the same sites as in Methanococcus cells. About 60% of the sequence of the in vitro RNA products was analyzed by dideoxyterminated primer extension and found to be identical with that of the precursors of tRNA(Val) and tRNA(Arg). This finding indicates that this RNA polymerase fraction both initiates and terminates transcription faithfully in vitro. After purification of a cell-free extract (S-100) of M. thermolithotrophicus by phosphocellulose chromatography, the endogenous RNA polymerase has lost its ability to transcribe the tRNA(Val) gene accurately. The activity directing specific expression of this template was reconstituted by the addition of a protein-fraction devoid of RNA polymerase activity. Thus, a transcription factor appears to be required for accurate cell-free expression of tRNA genes from M. vannielii.

At least two transcription factors, aTFB and aTFA, are required for accurate and faithful in vitro transcription of homologous templates in cell-free extracts from the methanogenic Archaeon Methanococcus thermolithotrophicus. We have recently shown that the function of aTFB can be replaced by eucaryal TATA-binding proteins. Here we demonstrate using template commitment experiments that promoter recognition in an Archaeon is mediated by transcription factors. The archaeal TATA box was identified as recognition site for binding of aTFB by gel shift analyses. aTFB binds also to the TATA box of adenovirus 2 major late promoter suggesting homology of eucaryal and archaeal TATA boxes. Our analyses provide evidence for a common molecular mechanism of transcription initiation by eucaryal RNA polymerases and archaeal RNA polymerase. They indicate also an evolutionary homology for aTFB and TBP.

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