Structure, Function, and Dynamics of Macro-molecular Complexes that Execute and Regulate Genome Function

执行和调节基因组功能的大分子复合物的结构、功能和动力学

• 批准号: 10594431
• 负责人: Effie Apostolou
• 金额: $ 253.6万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10594431
• 关键词: ATAC-seq; Advisory Committees; Affect; Affinity; Affinity Chromatography; Architecture; Automobile Driving; Base Pairing; Binding; Biological; Biological Assay; Biotinylation; Cell Separation; Cells; ChIP-seq; Chemicals; Chromatin; Complex; Consensus; Crosslinker; Cryoelectron Microscopy; Detection; Developmental Gene; Dimensions; Disease; Ensure; Epigenetic Process; Funding; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Health; Heterogeneity; Human; Individual; Inflammatory; Knowledge; Lead; Learning; Location; Macromolecular Complexes; Mass Spectrum Analysis; Measures; Membrane Proteins; Messenger RNA; Methods; Mitosis; Modeling; Molecular; Monitor; Pharmaceutical Preparations; Phosphorylation; Physiological; Polymerase; Positioning Attribute; Process; Proteins; Protocols documentation; Publications; RNA; RNA Polymerase II; Reagent; Regulation; Research; Research Personnel; Resolution; Role; Specificity; Streptavidin; Structure; Surface; Technology; Testing; Therapeutic Intervention; Transcriptional Regulation; aptamer; biological adaptation to stress; conflict resolution; crosslink; drug discovery; gene induction; genetic information; genome-wide; improved; inhibitor; insight; meetings; member; molecular mechanics; molecular modeling; novel; novel therapeutics; promoter; recruit; therapeutic development; therapeutic target; transcription factor

PROJECT SUMMARY/ABSTRACT The genetic information encoded in our genome is decoded and implemented via many multi-step processes, including the proper decoding by transcription. Transcription of genes into mRNA by RNA Polymerase II (Pol II) is a complex process that is precisely regulated both temporally and spatially at multiple steps by many large molecular complexes (LMCs). In the past, a number of these LMCs have been identified and their structural and functional role has been studied. Although we have learned a great deal about these LMCs at an individual level, how these LMCs interact and affect one another and Pol II at a more comprehensive level has yet to be achieved. In this project, we are proposing a multi-prong approach to define interactions and structures of LMCs, Pol II, and model transcription factors (TFs) in an unbiased way and, as much as possible, under native conditions. We will also evaluate the function of these specific interactions on the molecular mechanics of transcription and regulation in cells. To this end, we will utilize a novel GFP aptamer-based purification method to identify LMCs and TFs that associate with GFP-tagged Pol II and other critical LMCs. Purifications will be performed rapidly and under native conditions to ensure retention of physiological interactions, and the resulting complexes will be analyzed by both Mass Spectrometry and Cryo-EM to define the composition and structure of these LMCs at the highest depth and resolution possible. Crosslinking with novel protein-protein crosslinkers and subsequent MS analysis (XL-MS) will also be used to capture more transient LMC and TF interactions. In parallel, LMC-APEX2 fusions will be used to biotinylate nearby proteins and identify them by MS analysis following streptavidin purification. Additionally, we will define the location of distinctly modified Pol II complexes or Pol II associated with distinct LMCs at base-pair resolution along transcription units using our new PRO-IP-seq protocol. This information combined with the MS analysis provides a unique and dynamic view of Pol II’s phosphorylation status, composition, associations, and precise positioning along genes, and this information will be critical in deriving molecular models of transcription and its regulation. Previously known and newly identified LMCs and TFs that are deemed to have critical interactions will be perturbed by either RNA aptamer inhibitors or degron- tagging to tease apart their functional roles. The rapid expression RNA aptamers, which interfere with specific LMC interactions, and the rapid degradation of whole LMC subunits with degron technology will allow the detection of the immediate, “primary” roles of those interactions genome-wide using the high-resolution assays such as PRO-seq and ChIP-Exo. These assays will enable us to identify the specific functions of the key LMCs and their interactions at an unprecedented resolution and sensitivity. Overall, we expect to derive a much better and more complete understanding of the transcription cycle and its regulation. This will impact human health by identifying new therapeutic venues and possible lead drugs (RNA Aptamers), as misregulation of transcription has been observed in many disease conditions.

项目摘要/摘要 我们基因组中编码的遗传信息是通过许多多步骤过程解码和实现的， 包括通过转录进行适当的解码。通过RNA聚合酶II（POL II）将基因转录到mRNA中 是一个复杂的过程，在许多大型步骤上暂时和空间受到临时调节 分子复合物（LMC）。过去，许多这些LMC已被鉴定出来以及它们的结构和 功能作用已研究。尽管我们在个人层面上了解了有关这些LMC的大量知识，但 这些LMC如何相互作用并在更全面的水平上相互影响和影响Pol II。 在这个项目中，我们提出了一种多孔的方法来定义LMCS的相互作用和结构，Pol II， 和模型转录因子（TFS）以公正的方式，并在天然条件下尽可能多地进行。我们 还将评估这些特定相互作用对转录分子机制和 细胞中的调节。为此，我们将利用一种新型的GFP基于ATAMER的纯化方法来识别LMC 与GFP标记的Pol II和其他关键LMC相关的TF。纯化将迅速进行 在本地条件下以确保身体相互作用的保留，由此产生的复合物将是 通过质谱和冷冻EM分析以定义这些LMC在 最高深度和分辨率。与新型蛋白质 - 蛋白质交联和随后的MS交联 分析（XL-MS）也将用于捕获更多的瞬态LMC和TF相互作用。并行，LMC-APEX2 融合将用于生物素蛋白附近的蛋白质，并在链霉亲蛋白后通过MS分析识别它们 纯化。此外，我们将定义明显修改的Pol II配合物或Pol II相关的位置 使用我们的新Pro-IP-Seq协议，在沿转录单元沿转录单元的基本对分辨率下具有不同的LMC。这 信息与MS分析相结合，提供了Pol II磷酸化的独特而动态的视图 地位，构图，关联和沿基因的精确定位，此信息至关重要 得出转录的分子模型及其调节。以前已知和新确定的LMC和 RNA apatmer抑制剂或脱氧剂将扰动被认为具有关键相互作用的TF 标记以嘲笑其功能角色。快速表达RNA适体，干扰特定的 LMC相互作用以及整个LMC亚基与DEGRON技术的快速降解将允许 使用高分辨率测定法检测这些相互作用基因组的直接“主要”角色 例如Pro-Seq和Chip-Exo。这些测定将使我们能够确定关键LMC的特定功能 以及它们以前所未有的分辨率和灵敏度的相互作用。总体而言，我们希望得出更好 对转录周期及其调节的更完整了解。这将影响人类健康 识别新的治疗场所和可能的铅药（RNA APAMERS），因为转录的正调 在许多疾病状况中都观察到。