By using a recently developed in vitro transcription assay, the 16S/23S rRNA-encoding DNA promoter from the archaebacterium Sulfolobus sp. B12 was dissected by deletion and linker substitution mutagenesis. The analysis of 5' and 3' deletion mutants defmed a core promoter region between positions -38 and -2 containing all information for efficient and specific transcription. Further characterization of this region by linker substitution mutagenesis indicated two sequence elements important for promoter function-one located between positions -38 and -25 (distal promoter element) and the other one located between positions -11 and -2 (proximal promoter element). The distal promoter element encompassed the TATA-like "box A" element located approximately 26 nucleotides upstream of the majority of transcription start sites in archaebacteria (Archaeobacteria). Al mutations within this box A motif virtually abolished promoter function. Complete inactivation of the proximal promoter element was dependent on extensive mutagenesis; this element is not conserved between archaebacterial promoters except for a high A+T content in stable RNA gene promoters from Sulfolobus. Mutants containing insertions or deletions between the distal and proximal promoter elements were only slightly affected in their transcription efficiency but displayed a shift in their major initiation site, retaining an essentially fixed distance between the distal promoter element and the transcription start site. Thus, efficient transcription and start-site selection were dependent on a conserved TATA-like sequence centered approximately 26 nucleotides upstream of the initiation site, a situation unlike that of eubacterial promoters but resembling the core structure of most eukaryotic RNA polymerase II (and some RNA polymerase III) promoters. This rmding suggests a common evolutionary origin of these promoters consistent with the known similarities between archaebacterial and eukaryotic RNA polymerases.Based on molecular data, archaebacteria (Archaeobacteria) comprise a group of prokaryotic microorganisms phylogenetically distinct from eubacteria and eukaryotes (1-3). Many archaebacteria are characterized by extreme habitats believed to resemble the environmental conditions during the early evolution of life, leading to the suggestion that they represent an ancient group of organisms (1, 2).Transcription in archaebacteria has primarily been investigated on the level of DNA-dependent RNA polymerases. These studies indicate a single transcribing enzyme displaying a subunit complexity similar to the three eukaryotic nuclear enzymes (4). The characterization of archaebacterial transcription signals has been confined to nucleotide sequence comparisons, revealing two conserved sequence elements, a "box A" motif [consensus TTTA(A or T)A] centered approximately 26 nucleotides upstream of the transcription start site and a "box B" motif [consensus (A or T)TG(A or C)] containing the transcription start site on the central guanosine residue or on another pu...

The sequence requirements for specific and efficient transcription from the 16S/23S rRNA promoter of Sulfolobus shibatae were analysed by point mutations and by cassette mutations using an in vitro transcription system. The examination of the box A-containing distal promoter element (DPE) showed the great importance of the TA sequence in the center of box A for transcription efficiency and the influence of the sequence upstream of box A on determining the distance between the DPE and the start site. In most positions of box A, replacement of the wild type bases by adenines or thymines are less detrimental than replacements by cytosines or guanines. The effectiveness of the proximal promoter element (PPE) was not merely determined by its high A + T content but appeared to be directly related to its nucleotide sequence. At the start site a pyrimidine/purine (py/pu) sequence was necessary for unambiguous initiation as shown by analysis of mutants where the wild type start base was replaced. The sequence of box A optimal for promoter function in vitro is identical to the consensus of 84 mapped archaeal promoter sequences.

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We describe a cell-free transcription system for the archaebacterium Sulfolobus sp. B12 that specifically initiates transcription at the 5S rRNA-encoding DNA and the 16S/23S rRNA-encoding DNA promoters of the same species. With this crude extract system, specific initiation was absolutely dependent on the box A motif, a highly conserved promoter element in archaebacteria located approximately 25 base pairs upstream of transcription initiation sites. In vitro transcription of the rRNA genes by purified RNA polymerase, however, resulted in semi-specific, box A-independent initiation, indicating that factor(s) in the crude extract were necessary for the highly specific box A-dependent transcription. Fractionation of the cell-free extract by sucrose-gradient centrifugation resulted in the identification of a low molecular weight fraction complementing purified RNA polymerase to an extract-like specificity.Compared to the extensive knowledge about transcription in eubacteria and eukaryotes, little is known about RNA synthesis in archaebacteria, the third major phylogenetic group of organisms (1). This is largely due to the lack of specific in vitro transcription systems that would allow the determination of the single steps in the transcription process and the identification of the involved factors. A comparison between the transcribing enzymes of the three kingdoms reveals a multisubunit structure of the archaebacterial and the eukaryotic RNA polymerases that is considerably more complex than that of the eubacterial enzymes (2, 3). Immunochemical and amino acid sequence comparisons between the large subunits of DNA-dependent RNA polymerases support the hypothesis that archaebacterial RNA polymerases resemble their eukaryotic counterpart much more than eubacterial RNA polymerases (3). This similarity in the structure of the transcribing enzymes is paralleled by a similarity between archaebacterial promoters and the eukaryotic TATA-box containing polymerase II and polymerase III promoters (4,5), suggesting that the basic mechanism of transcription initiation might be similar in archaebacteria and eukaryotes.One approach to study transcription initiation of archaebacteria in more detail is the establishment of specific and efficient in vitro transcription systems. Here we describe a cell-free system derived from the archaebacterium Sulfolobus sp. B12, which specifically initiates the transcription of two rRNA genes at the known in vivo start sites. Specificity of initiation and promoter dependence were strikingly different between purified RNA polymerase and the crude extract system, indicating the requirement for one or more transcription factors. MATERIALS AND METHODSMaterials. Restriction endonucleases were obtained from BRL or from Boehringer Mannheim. S1 endonuclease, T4 polynucleotide kinase, and the Klenow fragment of Escherichia coli DNA polymerase I were purchased from Pharmacia; RNase-free DNase I was from Boehringer Mannheim, and Moloney murine leukemia virus reverse transcriptase was from BRL. All r...

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