Dynamic association of transcription initiation proteins with chromatin at single-molecule resolution in living yeast

细胞的生命是由基因组信息从DNA到RNA的受调控的转录所支配的 酶RNA聚合酶。转录调控是通过细胞中数百种蛋白质的作用实现的。 核，其身份和功能已从数十年的遗传和生化研究中阐明。 然而，人们对转录相关蛋白搜索和定位的时间尺度知之甚少。 活细胞中染色质的位置，以及染色质的修饰如何影响这种动态联系 建筑。这项提议旨在通过应用超分辨率荧光来解决这些问题 显微镜可以直接显示主要转录起始蛋白在单个细胞中的扩散运动。 在正常和改变的染色质结构状态下，芽殖酵母的分子分辨率。具体来说，我们 将检验这样的假设，即转录起始蛋白的染色质关联是以快速动力学发生的 而且染色质的重塑和修饰在调节转录蛋白质动力学中起着作用。 我们将使用活细胞单分子成像来监测转录起始蛋白的扩散行为。 在酵母核中具有高时空分辨率。这种‘体内生物化学’方法不同于并且是 补充了ChIP-Seq技术，该技术绘制了全基因组范围内的稳态占有率，但提供的信息很少 有关绑定动态的信息。我们将设计并从功能上验证编码的DNA结构 代表一般转录因子和主要序列特异性DNA结合的成分 转录因子融合到一个自我标记的蛋白标签(HaloTag)上，可以与细胞通透性的蛋白质发生共价反应 有机荧光团(Janelia Fluor)。单细胞荧光标记转录因子的活细胞成像 分子分辨率测量扩散系数，区分染色质结合和无染色质 并估计绑定人口的居留时间。此外，我们将使用有条件的耗尽 6个主要染色质重构体和组蛋白修饰物揭示转录迁移率的变化 启动蛋白，并告知几个扩散参数中的哪一个受染色质控制。这 条件突变遗传学和活细胞单分子成像的结合可能改变人们的理解 转录启动的动力学机制，并为酵母核的其他区域提供了一种新的途径 以及染色体生物学，包括DNA复制、修复和重组的研究。

The life of a cell is governed by the regulated transcription of genomic information from DNA to RNA by the enzyme RNA polymerase. Transcriptional control occurs through the actions of hundreds of proteins in the cell nucleus whose identities and functions have been elucidated from decades of genetic and biochemical studies. However, little is known about the timescales under which transcription-related proteins search for and target chromatin sites in living cells, and how such dynamic associations are influenced by modifications of chromatin architecture. This proposal aims to address these questions by applying super-resolution fluorescence microscopy to directly visualize the diffusive movements of major transcription initiation proteins at single- molecule resolution in budding yeast, under normal and altered states of chromatin architecture. Specifically, we will test the hypothesis that chromatin association of transcription initiation proteins occurs with rapid kinetics in live cells, and that chromatin remodeling and modification has a role in regulating transcription protein dynamics. We will use live-cell single-molecule imaging to monitor the diffusive behavior of transcription initiation proteins at high spatio-temporal resolution in the yeast nucleus. This `in vivo biochemistry' approach differs from and is complementary to ChIP-Seq techniques that map steady-state occupancies genome-wide but provide little information on binding dynamics. We will engineer and functionally validate DNA constructs encoding components representative of the general transcription factors and major sequence-specific DNA binding transcription factors fused to a self-labeling protein tag (HaloTag) that can react covalently with a cell-permeable organic fluorophore (Janelia Fluor). Live-cell imaging of fluorescently labeled transcription factors at single- molecule resolution measures diffusion coefficients, distinguishes between chromatin-bound and chromatin-free populations, and estimates residence times for the bound population. Further, we will use conditional depletion of 6 major chromatin remodelers and histone modifiers to reveal changes in the mobilities of transcription initiation proteins and inform which among several diffusive parameters are subject to chromatin controls. This combination of conditional mutant genetics and live-cell single-molecule imaging may transform understanding of the kinetic mechanisms for transcription initiation and offer a new approach to other areas of yeast nuclear and chromosome biology, including studies of DNA replication, repair, and recombination.