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: 10381452
• 负责人: Effie Apostolou
• 金额: $ 253.1万
• 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/10381452
• 关键词: ATAC-seq; Advisory Committees; Affect; Affinity; Architecture; Automobile Driving; Base Pairing; Binding; Biological; Biological Assay; Cells; ChIP-seq; Chemicals; Chromatin; Complex; Conflict (Psychology); Consensus; Crosslinker; Cryoelectron Microscopy; DNA Polymerase II; DNA-Directed RNA Polymerase; Detection; Developmental Gene; Dimensions; Disease; Electron Transport Complex III; Ensure; Epigenetic Process; Funding; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Goals; Health; Heterogeneity; Human; Individual; Inflammatory; Knowledge; Lead; Location; Macromolecular Complexes; Mass Spectrum Analysis; Measures; Membrane Proteins; Messenger RNA; Methods; Mitosis; Modeling; Molecular; Monitor; Pharmaceutical Preparations; Phosphorylation; Physiological; Positioning Attribute; Process; Proteins; Protocols documentation; Publications; RNA; Reagent; Regulation; Research; Research Personnel; Resolution; Role; Specificity; Streptavidin; Structure; Surface; Technology; Testing; Therapeutic Intervention; Transcriptional Regulation; aptamer; base; biological adaptation to stress; 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 编码在我们基因组中的遗传信息通过许多多步骤的过程被解码和实现， 包括通过转录进行正确的解码。RNA聚合酶II(POL II)将基因转录成mRNA 是一个复杂的过程，在时间和空间上都受到许多大的 分子络合物(LMC)。在过去，已经发现了一些这样的LMC，它们的结构和 对其功能作用进行了研究。虽然我们已经在个人层面上对这些LMC了解了很多， 这些小岛屿发展中国家如何在更全面的层面上相互作用和影响第二次人口普查尚未实现。 在这个项目中，我们提出了一个多管齐下的方法来定义LMC的相互作用和结构，POL II， 并以公正的方式，尽可能在自然条件下对转录因子(TF)进行建模。我们 还将评估这些特定相互作用在转录和分子机制上的功能 细胞内的调节。为此，我们将利用一种新的基于GFP适配子的纯化方法来鉴定LMC 以及与GFP标记的POL II和其他关键LMC相关联的TF。净化工作将迅速进行 并在自然条件下确保保留生理相互作用，得到的络合物将 用质谱仪和低温电子显微镜进行分析，以确定这些LMC的组成和结构 尽可能达到最高深度和分辨率。新型蛋白质-蛋白质交联剂及后续MS的研究 分析(XL-MS)还将用于捕获更多的瞬时LMC和TF相互作用。并行地，LMC-APEX2 融合将被用于生物素化附近的蛋白质，并通过链霉亲和素之后的MS分析来鉴定它们 净化。此外，我们将定义明显修改的POL II复合体或与POL II相关的POL II的位置 使用我们新的PRO-IP-SEQ协议，沿着转录单元在碱基对分辨率上具有不同的LMC。这 结合MS分析的信息提供了POL II磷酸化的独特和动态的视图 状态、组成、关联和基因的精确定位，这些信息将在 转录及其调控的分子模型的推导。以前已知和新发现的LMC和 被认为具有关键相互作用的转录因子将被RNA适体抑制剂或降解- 标记以梳理它们的功能角色。干扰特异性的快速表达RNA适配子 LMC相互作用，以及利用degron技术快速降解整个LMC亚基将允许 使用高分辨率分析检测这些相互作用在全基因组范围内的直接“主要”作用 如PRO-SEQ和CHIP-EXO。这些化验将使我们能够确定关键LMC的具体功能 以及它们以前所未有的分辨率和敏感度相互作用。总体而言，我们预计会有一个更好的 对转录周期及其调控有更全面的了解。这将通过以下方式影响人类健康 识别新的治疗场所和可能的先导药物(RNA适配子)，作为转录调控错误 在许多疾病情况下都观察到了。