title: Cell-free transcription of the nifH1 gene of Methanococcus thermolithotrophicus indicates that promoters of archaeal nif genes share basic features with the methanogen consensus promoter.
abstract: The nifH1 gene of Methanococcus thermolithotrophicus, which encodes the putative dinitrogenase reductase of an archaeon, was accurately transcribed in a homologous cell-free transcription system. Extracts of cells grown with N2 or ammonia as nitrogen source initiated transcription at the nifH1 promoter with similar efficiencies. We confirmed that cells grown under non-N2-fixing conditions do not contain significant amounts of nifH1-specific mRNA. The levels of cell-free transcription initiation at the nifH1 promoter were similar to those observed at a tRNA promoter. The DNA sequence from -40 to +5 relative to the initiator nucleotide of nifH1 mRNA contained all the information required for promoter activity. A mutational analysis of this section of DNA demonstrated that a TATA box at -25 and the TTGT motif (initiator element) at the transcription start site are essential for cell-free transcription. These elements are similar to the structural determinants of a known tRNA promoter of Methanococcus. Mutation of a sequence, showing homology to the bacterial NifA site, which overlaps the transcription start site, did not affect promoter activity. Hence, cell-free transcription of the Methanococcus nifH1 gene is independent of upstream activator elements and does not require alternate cis-acting sequences that differ from the methanogen consensus promoter. These findings suggest that the activation of nif promoters is brought about by fundamentally different mechanisms in Archaea and bacteria.
authors.affiliation: Lehrstuhl für Mikrobiologie, Universität Regensburg, FRG.
authors.forename: H P
authors.initials: HP
authors.lastname: Gohl
authors.forename: W
authors.initials: W
authors.lastname: Hausner
authors.forename: M
authors.initials: M
authors.lastname: Thomm
mesh_terms: D000641:Ammonia / Q000378:metabolism; D001483:Base Sequence; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D005798:Genes, Bacterial; D017017:Methanococcus / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000378:metabolism; D009586:Nitrogen Fixation / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D000641:Ammonia; D009584:Nitrogen