Ashley C, Lee J S
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
DNA Cell Biol. 2000 Apr;19(4):235-41. doi: 10.1089/104454900314500.
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.
多聚嘌呤 - 多聚嘧啶序列在真核生物中含量过高，其中许多有形成三链DNA的潜力。分子间三链体在双链DNA中分离但互补的多聚嘌呤 - 多聚嘧啶序列之间形成。先前使用含有一对设计为能够相互形成三链体的分离的多聚嘌呤 - 多聚嘧啶序列的环状质粒研究了分子间三链体（T环）。当带切口的质粒在低pH值下于精胺存在的情况下孵育时形成T环。当pH值升至8时，T环被由多链和单链区域组成的氢键结合的结所限制。本实验使用T环作为模型系统来研究分子间三链体形成对转录的影响。从琼脂糖凝胶上的条带中分离出T环以及对照开环、线性和超螺旋质粒形式。用分离出的质粒形式以及缺乏σ70的大肠杆菌RNA聚合酶全酶和核心酶进行转录测定。与对照质粒形式相比，T环形式的转录受到显著抑制。没有证据表明T环的单链区域促进了转录的非特异性起始。相反，T环结构根部氢键结合的结的多链成分抑制了转录。
