An Archaebacterial in Vitro Transcription System

Thomm, Michael; Frey, Gerhard; Hausner, Winfried; Brüdigam, Berit

doi:10.1007/978-1-4613-0613-9_27

Cited by 3 publications

(4 citation statements)

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“…Growth of methanogens and isolation of nucleic acids. Cultures of M. thermolithotrophicus and M. fervidus were grown anaerobically at 65 and 83°C, respectively (18,21,24). Nucleic acids were prepared as previously described from exponentially growing M. fervidus cells that were rapidly frozen in liquid N, and ruptured by grinding with a pestle and mortar (24).…”

Section: Methodsmentioning

confidence: 99%

“…Purification of RNAP and transcription factors. Components of the cell-free system from M. thennolithotrophicus were purified either (i) by phosphocellulose (PC) chromatography as described previously (21), in which case the 0.35 M KCI PC eluate contained the RNAP activity (0.25 mg/ml) and the 0.6 M KCI PC eluate contained a transcription factor (7), or (ii) by sequential S-100, DEAE-cellulose, heparin-cellulose, and Mono Q (fast protein liquid) chromatography, in which case the preparations obtained were -80% RNAP and were then combined with the PC transcription factor. M. thermolithotrophicus RNAP prepared by either procedure generated the same transcripts of the hmfB gene in vitro.…”

Section: Methodsmentioning

confidence: 99%

“…It was therefore somewhat of a surprise that although 16 of 17 bases in the TATA box region immediately upstream of the closely related hmtB gene, cloned from Methanobacterium thermoautotrophicum AH, were perfectly conserved (20), there was no duplicated region upstream of this gene. We have therefore investigated transcription of the hmfB gene both in vivo and in vitro and demonstrate here that although both TATA boxes associated with the cloned hmfB gene are recognized and used as promoters in vitro by RNAP from Methanococcus thermolithotrophicus (10,21), only one TATA box region is present and used as a promoter in vivo in M. fervidus. The duplication apparently occurred as an artifact during the original hmfB cloning.…”

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confidence: 92%

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Transcription in vivo and in vitro of the histone-encoding gene hmfB from the hyperthermophilic archaeon Methanothermus fervidus

et al. 1992

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Immediately upstream of the hmfB gene, in a DNA fragment cloned from Methanothermus fervidus, are two identical tandemly repeated copies of a 73-bp sequence that contain the sequence 5'TTTATATA, which conforms precisely to the consensus TATA box element proposed for methanogen promoters. By using this duplicated region as the template DNA and a cell-free transcription system derived from Methanococcus thermolithotrophicus, transcription in vitro was found to initiate at two identical sites 73 bp apart, each 25 bp downstream from a TATA box, thus providing strong evidence for the functional conservation of this transcriptional signal in two phylogenetically very diverse methanogens. Transcription of the hmfB gene in vivo in M. fervidus was found to occur at only one of these sites, and consistent with this observation, recloning and sequencing of this intergenic region after its amplification by the polymerase chain reaction demonstrated that the genome of M. fervidus contains only one copy of the 73-bp sequence upstream of the hmfB gene. Since the second copy of the 73-bp sequence, presumably generated artifactually during the original hmfB cloning, functioned equally well as a promoter in the M. thermolithotrophicus transcription system, all information needed by the heterologous RNA polymerase to initiate transcription accurately in vitro must be present within this sequence. The hmfB gene encodes HMf-2, one of the two subunits of HMf, an abundant DNA binding protein in M. fervidus which binds to DNA molecules in vitro, forming nucleosomelike structures. Cell-free transcription was inhibited by adding HMf or eucaryotic core histones at protein-to-DNA mass ratios of 0.3:1 and 1:1, respectively, whereas the archael histonelike protein HTa from Thermoplasma acidophilum inhibited transcription in vitro only at much higher protein-to-DNA mass ratios and the bacterial histonelike protein HU from Escherichia coli had no detectable effect on transcription.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 92%

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Transcription in vivo and in vitro of the histone-encoding gene hmfB from the hyperthermophilic archaeon Methanothermus fervidus

et al. 1992

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“…(A) DNA sequences upstream from archaeal tRNA/rRNA and 7S genes have been aligned to yield maximal homology, with TATA box at -25. This promoter element has been identified by footprinting and cell-free transcription experiments using purified components from Methanococcus (Thomm and Wich, 1988;Thomm et al, 1990;Hausner et al, 1991). The consensus octanucleotide (methanogens and halophiles) and hexanucleotide (Crenarchaeota) are boxed.…”

Section: S 7s16smentioning

confidence: 99%