Soukup G A, Maher J J
Department of Biochemistry and Molecular Biology, Mayo Foundation, Guggenheim 16, 200 First Street SW, Rochester, MN 55905, USA.
Nucleic Acids Res. 1998 Jun 1;26(11):2715-22. doi: 10.1093/nar/26.11.2715.
Oligonucleotide-directed triple helix formation offers a method for duplex DNA recognition, and has been proposed as an approach to the rational design of gene-specific repressors. Indeed, certain RNA and DNA oligonucleotides have previously been shown to bind duplex DNA and repress in vitro transcription by occluding the binding of transcription factors or RNA polymerase at target genes. While similar oligonucleotides have reportedly caused repression of target genes in cultured cells, physical evidence of triple helix formation in vivo is generally lacking. In the present study we wished to determine whether RNA transcripts could repress the activity of an Escherichia coli promoter in vivo by binding to the duplex promoter DNA. An in vivo genetic selection previously developed to identify DNA binding proteins was modified for this purpose. Using expression libraries encoding RNAs predisposed to forming triple helices with a DNA target site, we have selected RNA transcripts that confer survival to E.coli by disrupting transcriptional interference. Surprisingly, genetic and biochemical evidence shows that these RNAs do not form triple helices at the target promoter in vivo , despite the fact that they contain sequences capable of forming triple helices at the duplex DNA target in vitro . Rather, the selected RNAs appear to disrupt transcriptional interference via an antisense mechanism.
寡核苷酸定向三链螺旋的形成提供了一种识别双链DNA的方法，并已被提议作为一种合理设计基因特异性阻遏物的途径。事实上，某些RNA和DNA寡核苷酸此前已被证明能结合双链DNA，并通过在靶基因处阻断转录因子或RNA聚合酶的结合来抑制体外转录。虽然据报道类似的寡核苷酸能在培养细胞中抑制靶基因的表达，但体内三链螺旋形成的物理证据普遍缺乏。在本研究中，我们希望确定RNA转录本是否能通过与双链启动子DNA结合来在体内抑制大肠杆菌启动子的活性。为此，我们对之前开发的用于鉴定DNA结合蛋白的体内遗传筛选方法进行了改进。利用编码易于与DNA靶位点形成三链螺旋的RNA的表达文库，我们筛选出了通过破坏转录干扰赋予大肠杆菌生存能力的RNA转录本。令人惊讶的是，遗传和生化证据表明，这些RNA在体内靶启动子处并未形成三链螺旋，尽管它们含有在体外双链DNA靶位点能形成三链螺旋的序列。相反，所选的RNA似乎通过反义机制破坏转录干扰。
