In vitro transcription of two rRNA genes of the archaebacterium Sulfolobus sp. B12 indicates a factor requirement for specific initiation.

Abstract

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...

Mercury Inactivates Transcription and the Generalized Transcription Factor TFB in the Archaeon Sulfolobus solfataricus

“…All S. solfataricus cell extracts were prepared from cells harvested from cultures in mid-exponential phase having an OD 540 of 0.5. Cell extracts adjusted to either pH 6.0 or 8.0 were prepared as described previously (5,21). All reagents and plasticware used for preparation of cell extracts were made RNase free by treatment with diethylpyrocarbonate.…”

“…The amounts of pH 8.0 and pH 6.0 cell extracts used for in vitro transcription reactions were 50 and 400 g, respectively. The reactions were carried out at 60°C for 10 min for pH 8.0 cell extract, as described previously (21), and at 75°C for 10 min for pH 6.0 cell extract, as described previously (5). RNAs from in vitro transcription reactions were subjected to primer extension by generating cDNA using avian myeloblastosis virus reverse transcriptase (U.S. Biochemicals) or SuperScript II (Invitrogen) and [␥-32 P]ATP-labeled M13 primer (5Ј-AGCGGATAACAATTT CACACAGGAAACAGC-3Ј), as described previously (5).…”

“…The second approach investigated the effect of exogenous HgCl 2 addition to extracts prepared from untreated cells. Earlier studies (5) had shown that improved lacSp transcription occurred when transcription assays were conducted at pH 6.0 and 75°C rather than at pH 8.0 and 60°C as originally described for promoters of untranslated genes (21). Consequently, S. solfataricus cell extracts were prepared at both pH values according to the promoter under examination.…”

Enzymology of Base Excision Repair in the Hyperthermophilic Archaeon Pyrobaculum aerophilum

“…aerophilum Extracts and Purified Proteins-P. aerophilum cultures were grown in the laboratory of Jeffrey Miller (University of California, Los Angeles, CA), and cell-free extracts were prepared by Mahmud Shivji as described previously (24). The cell-free extract was supplemented with 1 mM phenylmethylsulfonyl fluoride and 1ϫ "Complete™, EDTA-free" (Roche Applied Science) protease inhibitor mixture and dialyzed overnight at 4°C against 2 liters of buffer containing 25 mM sodium phosphate, pH 8.0, 50 mM NaCl, 10% glycerol, 0.5 mM EDTA, and 2 mM DTT.…”

Two Transcription Factors Related with the Eucaryal Transcription Factors TATA-binding Protein and Transcription Factor IIB Direct Promoter Recognition by an Archaeal RNA Polymerase

“…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).…”