Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Mol Cell. 2013 Jun 6;50(5):711-22. doi: 10.1016/j.molcel.2013.05.016.
Pausing of RNA polymerase II (Pol II) 20-60 bp downstream of transcription start sites is a major checkpoint during transcription in animal cells. Mechanisms that control pausing are largely unknown. We developed permanganate-ChIP-seq to evaluate the state of Pol II at promoters throughout the Drosophila genome, and a biochemical system that reconstitutes promoter-proximal pausing to define pausing mechanisms. Stable open complexes of Pol II are largely absent from the transcription start sites of most mRNA genes but are present at snRNA genes and the highly transcribed heat shock genes following their induction. The location of the pause is influenced by the timing between when NELF loads onto Pol II and how fast Pol II escapes the promoter region. Our biochemical analysis reveals that the sequence-specific transcription factor, GAF, orchestrates efficient pausing by recruiting NELF to promoters before transcription initiation and by assisting in loading NELF onto Pol II after initiation.
暂停 RNA 聚合酶 II（Pol II）在转录起始位点下游 20-60 个碱基处是动物细胞中转录的一个主要检查点。控制暂停的机制在很大程度上是未知的。我们开发了高锰酸盐-ChIP-seq 来评估果蝇基因组中启动子处 Pol II 的状态，以及一种重新构建启动子近端暂停以定义暂停机制的生化系统。Pol II 的稳定开放复合物在大多数 mRNA 基因的转录起始位点基本不存在，但在 snRNA 基因和高度转录的热休克基因在诱导后存在。暂停的位置受 NELF 加载到 Pol II 的时间和 Pol II 从启动子区域逃逸的速度之间的时间差的影响。我们的生化分析表明，序列特异性转录因子 GAF 通过在转录起始前将 NELF 募集到启动子上来协调有效的暂停，并且在起始后协助 NELF 加载到 Pol II 上。
