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.

项目总结/摘要 编码在我们基因组中的遗传信息是通过许多多步骤过程解码和实现的， 包括通过转录的正确解码。通过RNA聚合酶II（Pol II）将基因转录成mRNA 这是一个复杂的过程，在时间和空间上都受到许多大的 分子复合物（LMCs）。在过去，已经确定了许多这样的LMC，并且它们的结构和 功能作用进行了研究。尽管我们已经在个体层面上对这些LMC有了很多了解， 这些LMC如何相互作用和相互影响以及Pol II在更全面层面上尚未实现。 在这个项目中，我们提出了一个多管齐下的方法来定义LMCs，Pol II， 和模型转录因子（TF）在一个公正的方式，并尽可能，在天然条件下。我们 还将评估这些特定相互作用对转录分子力学的作用， 细胞内的调节。为此，我们将利用一种新的基于GFP适体的纯化方法来鉴定LMCs 以及与GFP标记的Pol II和其他关键LMC相关的TF。净化将迅速进行 并在天然条件下，以确保保留生理相互作用，并且所得复合物将 通过质谱和Cryo-EM分析，以确定这些LMC的组成和结构， 最大的深度和分辨率。用新型蛋白质-蛋白质交联剂交联和随后的MS 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-APEX 2 融合体将用于生物素化附近的蛋白质，并在链霉亲和素后通过MS分析鉴定它们 洁净.此外，我们将定义明显修饰的Pol II复合物或Pol II相关的位置。 与不同的LMC在碱基对分辨率沿着转录单位使用我们的新的PRO-IP-seq协议。这 结合MS分析的信息提供了Pol II磷酸化的独特和动态视图 状态、组成、关联和沿基因的精确定位沿着，这些信息将是至关重要的， 推导转录及其调控的分子模型。以前已知和新发现的LMC， 被认为具有关键相互作用的TF将被RNA适体抑制剂或降解决定子干扰。 来区分它们的功能角色。快速表达RNA适体，其干扰特异性 LMC相互作用，以及用降解决定子技术快速降解整个LMC亚基，将使LMC的降解成为可能。 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 例如PRO-seq和ChIP-Exo。这些分析将使我们能够确定关键LMC的具体功能 以及它们之间的相互作用达到了前所未有的分辨率和灵敏度。总的来说，我们希望得到一个更好的 更全面地了解转录周期及其调控。这将影响人类健康， 确定新的治疗场所和可能的先导药物（RNA适体），作为转录的错误调节 在许多疾病中都可以观察到。