Affinity selection of DNA-binding protein complexes using mRNA display.
Affinity selection of DNA-binding protein complexes using mRNA display.
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DOI：
10.1093/nar/gnj025
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发表时间：
2006-02-14
影响因子：
14.9
通讯作者：
Yanagawa H
中科院分区：
文献类型：
--
作者：
Tateyama S;Horisawa K;Takashima H;Miyamoto-Sato E;Doi N;Yanagawa H
DNA-蛋白质相互作用的综合分析对于在全基因组水平上绘制转录调控网络图是重要的。在这里，我们提出了一个新的应用mRNA展示在体外选择的DNA结合蛋白异二聚体复合物。在改进的选择条件下，使用TPA响应元件（TRE）作为诱饵DNA，已知的相互作用c-fos和c-jun同时富集约100倍，从模型库（c-fos，c-jun和gst基因的1：1：20 000的混合物）后，一轮的选择。此外，从小鼠脑poly A+ RNA构建的mRNA展示文库中，经过6轮筛选，几乎所有的AP-1家族基因（包括c-jun、c-fos、junD、junB、atf 2和b-atf）都被成功地筛选到。这些结果表明，mRNA展示选择系统可以在单个实验中识别多种DNA结合蛋白复合物。由于几乎所有的转录因子形成异源寡聚复合物与其靶DNA结合，这种方法应该是最有用的DNA结合转录因子复合物的搜索。
Comprehensive analysis of DNA–protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro selection of DNA-binding protein heterodimeric complexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultaneously enriched about 100-fold from a model library (a 1:1:20 000 mixture of c-fos, c-jun and gst genes) after one round of selection. Furthermore, almost all kinds of the AP-1 family genes including c-jun, c-fos, junD, junB, atf2 and b-atf were successfully selected from an mRNA display library constructed from a mouse brain poly A+ RNA after six rounds of selection. These results indicate that the mRNA display selection system can identify a variety of DNA-binding protein complexes in a single experiment. Since almost all transcription factors form heterooligomeric complexes to bind with their target DNA, this method should be most useful to search for DNA-binding transcription factor complexes.