A triplex-mediated knot between separated polypurine-polypyrimidine tracts in circular DNA blocks transcription by Escherichia coli RNA polymerase

Abstract

Polypurine-polypyrimidine tracts are overrepresented in eukaryotes and many have the potential to form triplex DNA. Transmolecular triplexes form between separated but complementary polypurine-polypyrimidine tracts in duplex DNA. Transmolecular triplexes (T-loops) were studied previously using a circular plasmid containing a pair of separated polypurine-polypyrimidine tracts designed to able to form a triplex with each other. T-Loops formed when the nicked plasmid was incubated at low pH in the presence of spermine. When the pH was raised to 8, the T-loops were constrained by a hydrogen-bonded knot composed of multistranded and single-stranded regions. The present experiments used T-loops as a model system to investigate the influence of transmolecular triplex formation on transcription. T-Loops and control open circular, linear, and supercoiled plasmid forms were isolated from bands on agarose gels. Transcription assays were carried out with the isolated plasmid forms and Escherichia coli RNA polymerase holoenzyme and the core enzyme, which lacked sigma70. Transcription was significantly inhibited in T-loop forms compared with control plasmid forms. There was no evidence that the single-stranded regions of T-loops facilitated nonspecific initiation of transcription. Instead, the multistranded component of the hydrogen-bonded knot at the root of the T-loop structure inhibited transcription.