Holstege F C, Timmers H T
Laboratory for Physiological Chemistry, Utrecht University, The Netherlands.
Methods. 1997 Jul;12(3):203-11. doi: 10.1006/meth.1997.0472.
Open complex formation precedes initiation of transcription by RNA polymerases. In the analysis of transcription initiation from eukaryotic class II promoters, we have used promoter DNA structures that represent intermediates in open complex formation. We describe the preparation and isolation of heteroduplex promoter fragments. Probes containing these DNA structures have a general application in the study of proteins binding to junctions of double- and single-stranded DNA. Such proteins play important roles not only in the regulation of RNA synthesis but also in processes like repair, replication, and recombination of DNA. In addition, a protocol is provided for a rapid and quantitative assay for open complexes and transcription bubbles using potassium permanganate as a chemical probe for single-stranded regions in DNA.
开放复合物的形成先于RNA聚合酶启动转录。在分析真核生物II类启动子的转录起始时，我们使用了代表开放复合物形成中间体的启动子DNA结构。我们描述了异源双链启动子片段的制备和分离。含有这些DNA结构的探针在研究与双链和单链DNA交界处结合的蛋白质方面具有广泛应用。这类蛋白质不仅在RNA合成的调控中发挥重要作用，而且在DNA修复、复制和重组等过程中也发挥重要作用。此外，还提供了一种使用高锰酸钾作为DNA单链区域化学探针的快速定量检测开放复合物和转录泡的方法。
