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首页> 外文期刊>Biochemistry >Inhibition of transcription elongation in the HER-2eu coding sequence by triplex-directed covalent modification of the template strand.
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Inhibition of transcription elongation in the HER-2eu coding sequence by triplex-directed covalent modification of the template strand.

机译：通过模板链的三重定向共价修饰来抑制HER-2 / neu编码序列中的转录延长。

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摘要

Triplex formation may be of potential utility to inhibit the expression of individual genes. We describe the formation of a triple helix in the coding sequence of the HER-2eu gene. In vitro transcription analysis in the presence and absence of triplex formation demonstrates that an unmodified DNA triplex-forming oligonucleotide is incapable of inhibiting RNA polymerase elongation. Triplex formation by an oligonucleotide-psoralen conjugate was used to form a covalent photoadduct with a thymine on the nontemplate strand of the HER-2eu gene. In the native HER-2eu gene, covalent attachment of the triplex-forming oligonucleotide to the nontemplate strand did not prevent RNA polymerase elongation. Using HER-2eu point mutants that would place the target thymine on the template strand, we demonstrated that covalent modification of the template strand was necessary to inhibit RNA polymerase elongation. Based on these data, we synthesized oligonucleotide-alkylator conjugates that would react with a specific guanine residue on the template strand of the HER-2eu coding sequence. The oligonucleotide-alkylator conjugates inhibited transcription elongation by T7 RNA polymerase and eukaryotic RNA polymerase II from a HeLa nuclear extract. These studies demonstrate the successful application of triplex-forming oligonucleotide-alkylator conjugates to inhibit transcription elongation in the HER-2eu gene, and show that covalent modification of the DNA strand used as the transcription template is necessary to prevent RNA polymerase elongation.
机译：三链体的形成可能具有抑制单个基因表达的潜在用途。我们描述了HER-2 / neu基因的编码序列中三螺旋的形成。在存在和不存在三链体形成的情况下的体外转录分析表明，未修饰的DNA形成三链体的寡核苷酸不能抑制RNA聚合酶的延长。通过寡核苷酸-补骨脂素缀合物形成的三链体被用于在HER-2 / neu基因的非模板链上与胸腺嘧啶形成共价光加合物。在天然HER-2 / neu基因中，三链体形成寡核苷酸与非模板链的共价结合不会阻止RNA聚合酶的延长。使用将靶胸腺嘧啶置于模板链上的HER-2 / neu点突变体，我们证明了模板链的共价修饰对于抑制RNA聚合酶的延长是必要的。根据这些数据，我们合成了可与HER-2 / neu编码序列的模板链上的特定鸟嘌呤残基反应的寡核苷酸-烷基化缀合物。寡核苷酸-烷化剂缀合物通过HeLa核提取物中的T7 RNA聚合酶和真核RNA聚合酶II抑制转录延伸。这些研究证明了形成三链体的寡核苷酸-烷基化缀合物成功地抑制了HER-2 / neu基因中的转录延长，并显示了用作转录模板的DNA链的共价修饰对于防止RNA聚合酶的延长是必要的。

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