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.

项目概要/摘要 我们基因组中编码的遗传信息通过许多多步骤过程进行解码和实现， 包括通过转录进行正确的解码。 RNA 聚合酶 II (Pol II) 将基因转录为 mRNA 是一个复杂的过程，由许多大型机构在时间和空间上分多个步骤进行精确调节 分子复合物（LMC）。过去，已经确定了许多此类 LMC，其结构和 的功能作用进行了研究。尽管我们在个体层面上对这些 LMC 有了很多了解， 这些LMC如何在更全面的层面上相互作用和影响彼此以及Pol II尚未实现。 在这个项目中，我们提出了一种多管齐下的方法来定义 LMC 的相互作用和结构，Pol II， 并尽可能在天然条件下以公正的方式对转录因子 (TF) 进行建模。我们 还将评估这些特定相互作用对转录和分子机制的作用 细胞内的调节。为此，我们将利用一种新型的基于 GFP 适体的纯化方法来鉴定 LMC 以及与 GFP 标记的 Pol II 和其他关键 LMC 相关的 TF。纯化将快速进行 并在自然条件下确保生理相互作用的保留，所得复合物将 通过质谱和冷冻电镜进行分析，以确定这些 LMC 的组成和结构 可能的最高深度和分辨率。使用新型蛋白质-蛋白质交联剂进行交联以及随后的 MS 分析 (XL-MS) 还将用于捕获更多瞬态 LMC 和 TF 相互作用。同时，LMC-APEX2 融合体将用于对附近的蛋白质进行生物素化，并通过链霉亲和素后的 MS 分析来识别它们 纯化。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 使用我们新的 PRO-IP-seq 协议，沿着转录单元以碱基对分辨率获得不同的 LMC。这 信息与 MS 分析相结合，提供了 Pol II 磷酸化的独特动态视图 沿着基因的状态、组成、关联和精确定位，这些信息对于 推导转录及其调控的分子模型。先前已知的和新确定的 LMC 以及 被认为具有关键相互作用的 TF 将受到 RNA 适体抑制剂或降解决定子的干扰 标记以区分它们的功能角色。快速表达的RNA适体，干扰特定的 LMC 相互作用以及利用降解决定子技术快速降解整个 LMC 亚基将使 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。这些测定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灵敏度进行交互。总体而言，我们期望获得更好的结果 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 适体），作为转录的错误调节 已在许多疾病状况中观察到。