细菌染色体DNA由称为细菌染色质蛋白的蛋白质构成和压缩(即,类核苷相关蛋白或NAP)。DNA依赖性RNA聚合酶(RNAP)必须经常与细菌染色质蛋白相互作用,因为它们通常在整个基因组范围内结合DNA。在某些情况下,RNAP必须克服细菌染色质蛋白对转录的影响。大肠杆菌中影响转录的一个关键细菌染色质蛋白是组蛋白样核苷酸结构蛋白,H-NS.H-NS与DNA结合并形成核蛋白丝。为了研究H-NS丝对RNAP伸长率的影响,我们开发了一种体外转录测定法来监测RNAP在H-NS结合的DNA模板上的进展。在这种方法中,RNAP的启动和延伸通过在仅有三个核糖核苷三磷酸(rNTP)存在的情况下首先启动转录以停止启动子下游的延伸而分离。在通过添加第四个NTP重新开始伸长之前,在DNA上形成H-NS纤丝,使得转录物延伸发生在H-NS核蛋白纤丝模板上。这里,我们提供了通过H-NS丝进行体外转录的详细方案,分析转录产物,并通过电泳迁移率变化测定法(EMSA)观察DNA上H-NS丝的形成。这些方法能够深入了解H-NS如何影响RNAP转录物延伸,并提供确定其他细菌染色质蛋白对RNAP延伸的影响的起点。
Bacterial chromosomal DNA is structured and compacted by proteins known as bacterial chromatin proteins (i.e., nucleoid-associated proteins or NAPs). DNA-dependent RNA polymerase (RNAP) must frequently interact with bacterial chromatin proteins because they often bind DNA genome-wide. In some cases, RNAP must overcome barriers bacterial chromatin proteins impose on transcription. One key bacterial chromatin protein in Escherichia coli that influences transcription is the histone-like nucleoid structuring protein, H-NS. H-NS binds to DNA and forms nucleoprotein filaments. To investigate the effect of H-NS filaments on RNAP elongation, we developed an in vitro transcription assay to monitor RNAP progression on a DNA template bound by H-NS. In this method, initiation and elongation by RNAP are uncoupled by first initiating transcription in the presence of only three ribonucleoside triphosphates (rNTPs) to halt elongation just downstream of the promoter. Before elongation is restarted by addition of the fourth NTP, an H-NS filament is formed on the DNA so that transcript elongation occurs on an H-NS nucleoprotein filament template. Here, we provide detailed protocols for performing in vitro transcription through H-NS filaments, analysis of the transcription products, and visualization of H-NS filament formation on DNA by electrophoretic mobility shift assay (EMSA). These methods enable insight into how H-NS affects RNAP transcript elongation and provide a starting point to determine effects of other bacterial chromatin proteins on RNAP elongation.