Institute for Genetics, University of Cologne, Cologne, Germany.
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA.
Methods Mol Biol. 2024;2819:225-240. doi: 10.1007/978-1-0716-3930-6_11.
Bacterial nucleoid-associated proteins are important factors in regulation of transcription, in nucleoid structuring, and in homeostasis of DNA supercoiling. Vice versa, transcription influences DNA supercoiling and can affect DNA binding of nucleoid-associated proteins (NAPs) such as H-NS in Escherichia coli. Here we describe genetic tools to study the interplay between transcription and nucleoid-associated proteins in E. coli. These methods include construction of genomic and plasmidic transcriptional and translational lacZ reporter gene fusions to study regulation of promoters; insertion of promoter cassettes to drive transcription into a locus of interest in the genome, for example, an H-NS-bound locus; and construction of isogenic hns and stpA mutants and precautions in doing so.
细菌核小体相关蛋白是转录调控、核小体结构和 DNA 超螺旋稳态的重要因素。反之，转录会影响 DNA 超螺旋，并可能影响 DNA 与核小体相关蛋白（NAPs）如大肠杆菌中的 H-NS 的结合。本文描述了研究大肠杆菌中转录与核小体相关蛋白相互作用的遗传工具。这些方法包括构建基因组和质粒转录和翻译 lacZ 报告基因融合，以研究启动子的调控；插入启动子盒以驱动转录进入基因组中的感兴趣基因座，例如 H-NS 结合基因座；以及构建同种 hns 和 stpA 突变体及其注意事项。
