A triplex-mediated knot between separated polypurine-polypyrimidine tracts in circular DNA blocks transcription by Escherichia coli RNA polymerase
A triplex-mediated knot between separated polypurine-polypyrimidine tracts in circular DNA blocks transcription by Escherichia coli RNA polymerase
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DOI：
10.1089/104454900314500
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发表时间：
2000-04-01
影响因子：
3.1
通讯作者：
Lee, JS
中科院分区：
文献类型：
--
作者：
Ashley, C;Lee, JS
多聚嘌呤-多聚嘧啶束在真核生物中过多存在，并且许多具有形成三聚体DNA的潜力，双分子三链体在双链体DNA中分离但互补的多聚嘌呤-多聚嘧啶束之间形成，双分子三链体（T环）先前使用含有一对分离的多聚嘌呤-多聚嘧啶束的环状质粒进行了研究，所述一对分离的多聚嘌呤-多聚嘧啶束被设计成能够彼此形成三聚体，当在精胺存在下在低pH下孵育带切口的质粒时，形成T环。当pH升高到8时，T环被多链和单链区域形成的氢键结所束缚。本实验以T环为模型系统，研究了跨分子三聚体形成对转录的影响。从琼脂糖凝胶上的条带中分离T-环和对照开环、线性和超螺旋质粒形式。用分离的质粒形式和大肠杆菌RNA聚合酶全酶和缺乏σ（70）的核心酶进行转录测定。与对照质粒形式相比，T环形式的转录被显著抑制。没有证据表明T环的单链区域促进了非特异性转录起始。相反，位于T环结构根部的氢键结的多链成分抑制了转录。
Polypurine-polypyrimidine tracts are overrepresented in eukaryotes and many have the potential to form tripler 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 tripler 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 multi-stranded and single-stranded regions, The present experiments used T-loops as a model system to investigate the influence of transmolecular tripler 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 sigma(70), 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.