Meiji University, School of Agriculture, Kawasaki, Kanagawa, Japan.
Research Center for Advanced Science and technology, Division of Gene Research, Shinshu University, Ueda, Nagano, Japan.
Methods Mol Biol. 2024;2819:77-102. doi: 10.1007/978-1-0716-3930-6_5.
The genome of Escherichia coli K-12 is transcribed by a single species of RNA polymerase. The selectivity of transcriptional targets is determined via interaction with one of seven species of the sigma subunit and a total of approximately 300 species of transcription factor (TFs). For comprehensive identification of the regulatory targets of these two groups of regulatory proteins on the genome, we developed an in vitro approach, "Genomic SELEX" (gSELEX) screening. Here we describe a detailed protocol of the gSELEX screening system, which uses purified regulatory proteins and fragments of genomic DNA from E. coli. Moreover, we describe methods and examples of results using cell-free synthetic proteins.
大肠杆菌 K-12 的基因组由一种 RNA 聚合酶转录。转录靶标的选择性通过与 7 种 σ 亚基之一和总共约 300 种转录因子（TFs）的相互作用来确定。为了全面鉴定这两组调节蛋白在基因组上的调节靶标，我们开发了一种体外方法，即“基因组 SELEX”（gSELEX）筛选。本文详细描述了 gSELEX 筛选系统的方案，该系统使用来自大肠杆菌的纯化调节蛋白和基因组 DNA 片段。此外，我们还描述了使用无细胞合成蛋白的方法和结果示例。
