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的受调控转录控制的， RNA聚合酶。转录控制通过细胞中数百种蛋白质的作用发生 细胞核的身份和功能已从几十年的遗传和生物化学研究中阐明。 然而，人们对转录相关蛋白寻找和靶向的时间尺度知之甚少。 活细胞中的染色质位点，以及染色质修饰如何影响这种动态关联 架构该提案旨在通过应用超分辨率荧光来解决这些问题 显微镜直接可视化的扩散运动的主要转录起始蛋白在单一的， 分子分辨率在芽殖酵母，在正常和染色质结构的改变状态。我们特别 将测试这一假设，即染色质协会的转录起始蛋白发生快速动力学， 活细胞，染色质重塑和修饰在调节转录蛋白动力学中起作用。 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 在酵母细胞核中的高时空分辨率。这种“体内生物化学”方法不同于 与ChIP-Seq技术互补，ChIP-Seq技术在全基因组范围内定位稳态占位基因， 关于绑定动力学的信息。我们将设计和功能验证DNA结构编码 代表一般转录因子和主要序列特异性DNA结合的组分 融合到自标记蛋白标签（HaloTag）的转录因子，所述自标记蛋白标签可以与细胞可渗透的 有机荧光团（Janelia Fluor）。荧光标记的转录因子在单细胞水平的活细胞成像 分子分辨率测量扩散系数，区分染色质结合和无染色质 人口，并估计滞留时间的约束人口。此外，我们将使用条件耗尽 6个主要的染色质重塑和组蛋白修饰剂，以揭示转录迁移率的变化 起始蛋白质和信息，其中几个扩散参数受到染色质控制。这 条件突变遗传学和活细胞单分子成像的结合可能会改变对 的转录起始的动力学机制，并提供了一个新的途径，其他领域的酵母核 和染色体生物学，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.