A transcriptionally active plasmid-protein complex isolated from Escherichia coli

A stable transcriptionally active plasmid-protein complex has been isolated in high yield from Escherichia coli containing the thermally-inducible plasmid pKN 402A. The complexes which have a protein/DNA weight ratio of approx. 1 contain more than 11 polypeptide species. The weight percents of the three known proteins in the complex H1, RNA polymerase and HU, are 23, 23 and 5%, respectively. In vitro RNA synthesis by this complex proceeds for several hours and is inhibited by rifampicin and actinomycin to 33 and 98%, respectively, suggesting that most of the observed nucleotide incorporation is due to elongation of preinitiated RNA chains. Exogenous E. coli RNA polymerase but not exogenous DNA stimulates the in vitro transcription indicating that RNA polymerase is limiting and binds tightly to the plasmid. Stimulation of the in vitro transcription by the addition of exogenous E. coli core polymerase suggests that sigma subunit may be released in the RNA synthesis. This transcriptionally active complex should prove to be useful to study the mechanism of transcription and regulation in vivo.