Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters
Genome-wide Single-Molecule Footprinting Reveals High RNA Polymerase II Turnover at Paused Promoters
复制标题
DOI：
10.1016/j.molcel.2017.06.027
复制
发表时间：
2017-08-03
期刊：
影响因子：
16
通讯作者：
Schubeler, Dirk
中科院分区：
文献类型：
--
作者：
Krebs, Arnaud R.;Imanci, Dilek;Schubeler, Dirk
转录起始需要染色质开放，随后是起始前复合物形成和RNA聚合酶II募集。随后的聚合酶延伸需要额外的信号，导致起始位点下游的停留时间增加，这种现象称为暂停。在这里，我们利用单分子足迹来量化整个果蝇基因组体内启动的不同步骤。这确定了启动子结构对核小体占据相关的启动动力学的影响。此外，转录起始扰动揭示了一个意想不到的高营业额的聚合酶在暂停启动子的观察证实在新生RNA的水平。这些观察结果表明，延伸的缺乏主要是由过早终止而不是由稳定的聚合酶停滞引起的。为了支持这种非进行性模型，我们观察到暂停的热休克启动子的诱导依赖于连续启动。我们的研究提供了一个框架，以量化蛋白质结合在单分子分辨率和完善的概念，转录暂停。
Transcription initiation entails chromatin opening followed by pre-initiation complex formation and RNA polymerase II recruitment. Subsequent polymerase elongation requires additional signals, resulting in increased residence time downstream of the start site, a phenomenon referred to as pausing. Here, we harnessed single-molecule footprinting to quantify distinct steps of initiation in vivo throughout the Drosophila genome. This identifies the impact of promoter structure on initiation dynamics in relation to nucleosomal occupancy. Additionally, perturbation of transcriptional initiation reveals an unexpectedly high turnover of polymerases at paused promoters-an observation confirmed at the level of nascent RNAs. These observations argue that absence of elongation is largely caused by premature termination rather than by stable polymerase stalling. In support of this non-processive model, we observe that induction of the paused heat shock promoter depends on continuous initiation. Our study provides a framework to quantify protein binding at single-molecule resolution and refines concepts of transcriptional pausing.