[Col E1 DNA transcription in Escherichia coli RNA-polymerase system in vitro]

The ColEI plasmid DNA transcription in E. coli RNA-polymerase system in vitro was studied. Three RNA types: of 110-140 residues long, 1700 residues long and of the length similar to that of DNA-template were synthesized in the reaction, containing 10 mM MgCl2, 50 mM KCl and 15% glycerol. The transcription was shown to be asymmetric, the DNA H-chain being transcribed. The ColEI DNA region transcribed in vitro was located by the blotting techniques. This region comprised most of the ColEI genome except the colicin EI gene. The addition of mitomycin C to the reaction mixture caused only a little stimulation of colicin EI gene transcription. Four fragments of HaeIII digest of ColEI DNA were shown to have affinity to RNA-polymerase in the absence of NTPs and two fragments to have it in the presence of 3 NTPs (in the RNA initiation conditions). These two fragments seem to contain two strong promoters. One of them is in the HaeIIIA fragment, where the colicin EI gene origin is situated, and another one is the HaeIIIB fragment (in the immunity region).