Transcription in methanogens. Evidence for specific in vitro transcription of the purified DNA-dependent RNA polymerase of Methanococcus thermolithotrophicus.
The purification of the DNA-dependent RNA polymerase of Methanococcus thermolithotrophicus is described. As the first step of purification the endogenous template was removed from the enzyme by hydrophobic interaction chromatography. The purified enzyme consists of seven components with different molecular masses. Transcription studies on T7 DNA and the recombinant plasmid pMV15, containing rRNA genes of Methanococcus vannielii, revealed that only the methanogen DNA is transcribed specifically, indicating a principal structural difference between archaebacterial and eubacterial promoters. This could be shown both by analysis of ternary transcription complexes and Southern hybridization. The site of initiation was found within a restriction fragment harbouring the first 390 nucleotides of the sequence coding for mature 16S rRNA and 1100 base pairs of upstream sequences. The specific initiation on this fragment strongly suggests that the enzyme can start in vitro transcription from the promoter(s) of rRNA synthesis.