Structural studies of eukaryotic transcription

真核转录的结构研究

基本信息

  • 批准号:
    10673042
  • 负责人:
  • 金额:
    $ 48.69万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2003
  • 资助国家:
    美国
  • 起止时间:
    2003-09-01 至 2026-08-31
  • 项目状态:
    未结题

项目摘要

ABSTRACT Cellular differentiation, development and homeostasis depend on regulation of gene expression, which is largely focused on the DNA transcription initiation process. During transcription initiation, Mediator, a large multi-protein complex conserved throughout eukaryotes, conveys regulatory signals to RNA polymerase II (RNAPII), the enzyme responsible for transcription of all protein-coding genes. Mediator includes 25-30 different polypeptides (depending on the specific organism) organized into Head, Middle and Tail modules, plus a dissociable kinase module (CKM) that includes the Cdk8 kinase, the only catalytically-active Mediator subunit. Mediator conformational rearrangements that stabilize preinitiation complex (PIC) components have explained the effect of Mediator on basal transcription. However, conformational rearrangements alone are insufficient to explain the response of Mediator to transcription factors (TFs) that enables transcription activation and repression. Here we propose biochemical, functional and cryo-EM studies of mammalian Mediator (mMED) that build on our previous work and explore the significance of mMED’s antagonistic interaction with the CKM and with MED26, a metazoan-specific, dissociable mMED subunit closely linked to modulation of mMED–RNAPII interaction. The CKM and MED26 interact with Mediator around a Head-Middle module interface (the CTD-binding gap) where RNAPII interaction is initiated by binding of the carboxy- terminal domain of the largest RNAPII subunit (the CTD). CKM-bound (CKM-mMED) and MED26-bound (MED26-mMED) forms of mMED were independently identified by various research groups shortly after Mediator’s discovery and we propose to test a mMED activation mechanism based on CKM-mMED to MED26- mMED interconversion that we hypothesize controls mMED interaction with RNAPII and PIC formation. In Aim1 we will Investigate the connection between CKM – MED26 antagonism and Mediator activation. We posit that the crux of the mMED activation mechanism is control of the initial CTD-dependent mMED– RNAPII interaction by antagonistic effects of the CKM (limits RNAPII interaction) and MED26 (required for RNAPII interaction) at the CTD-binding gap. We will use in vitro and in vivo approaches including biochemical, functional and genomic analyses to understand modulation of mMED interaction with RNAPII and its effects on transcription initiation. These studies will test a proposed activation mechanism that would explain the significance of mMED subpopulations with opposite functional effects and test whether interconversion between mMED forms can explain mMED activation. In Aim 2 we will determine the structural underpinnings that enable regulation of Mediator-RNAPII interaction. We postulate that TF targeting of CKM-mMED and subsequent effects on MED26 and CTD interaction at the CTD-binding gap are enabled by mMED structural rearrangements or changes in mMED conformational dynamics. Structural analysis of well-defined intermediate steps will reveal how mMED’s structure enables activation. We will use cryo-EM to determine near-atomic resolution maps of various intermediates and use state-of-the-art image analysis approaches to understand their conformational and interaction dynamics. These studies will reveal structural factors that underpin primary (initial CTD-dependent interaction with RNAPII) and subsequent (further modulation of RNAPII interaction and PIC assembly) aspects of the mMED activation mechanism. In Aim 3 we will investigate long-range structural rearrangements in mMED that enable mMED activation by TF binding to Tail module subunits. We believe that changes in the composition, structure or conformational dynamics of the Tail module triggered by interaction with TFs can be communicated along the mMED structure to the CTD-binding gap, allowing mMED to respond to a variety of regulatory signals through the same fundamental activation mechanism. We will use cryo-EM, image analysis and biochemistry to understand how signals from TF binding to various mMED Tail subunits converge to control interaction of mMED with RNAPII. The studies described in this aim will test the generality of the proposed mMED activation mechanism and reveal how the mMED structure can integrate signals from a variety of TFs that target different mMED subunits. The studies proposed in this application are both conceptually and technically innovative. We will test a novel model for regulation of activated transcription initiation based on interconversion between “repressed” and “activated” forms of mMED by applying a multi-disciplinary approach combining state-of-the-art molecular biology, genomics, bioinformatics and cryo-EM/image analysis techniques to understand early steps of mammalian PIC assembly and transcription initiation. We will combine in vitro studies allowing examination of individual aspects of the proposed mechanism with in vivo studies to verify biological significance and functional consequences. The use of state-of-the-art cryo-EM and image analysis will allow us to build on prior accomplishments and arrive at a detailed structural understanding of the way in which the intricate mMED structure enables activated transcription regulation.
摘要 细胞的分化、发育和动态平衡依赖于基因表达的调节,这是 主要集中在DNA转录起始过程。在转录启动过程中,一个大的 真核生物中保守的多蛋白复合体向RNA聚合酶II传递调控信号 (RNAPII),负责转录所有蛋白质编码基因的酶。调解员包括25-30人 不同的多肽(取决于特定的生物体)被组织成头部、中间和尾部模块, 外加一个包括CDK8激酶的可分离激酶模块(CKM),CDK8是唯一具有催化活性的介体 亚单位。稳定预引发复合体(PIC)组分的介体构象重排 解释了Mediator对基础转录的影响。然而,仅构象重排就是 不足以解释介体对转录因子(TF)的反应 激活和压抑。在这里,我们提出了哺乳动物的生化、功能和冷冻-EM研究 调解人(MMED),建立在我们之前工作的基础上,并探索MMED对抗的意义 与CKM和MED26的相互作用,MED26是一种后生动物特有的、可解离的MMEd亚单位,与 MMEd-RNAPII相互作用的调制。CKM和MED26在头部-中部周围与调解器交互 模块界面(CTD结合间隙),其中RNAPII相互作用是通过结合羧基- 最大的RNAPII亚单位(CTD)的终端域。CKm-Bound(CKM-MMEd)和MED26-Bound (MED26-MMEd)形式的MMED在不久后被各个研究小组独立鉴定 Mediator的发现,我们建议测试一种基于CKM-MMEd到MED26的MMEd激活机制- 我们假设的MMEd相互转换控制着MMEd与RNAPII和PIC的形成。 在Aim1中,我们将研究CKM-MED26拮抗与介体激活之间的关系。 我们认为,MMEd激活机制的关键是控制初始的CTD依赖的MMEd- RNAPII相互作用通过CKM(限制RNAPII相互作用)和MED26的拮抗作用(需要 RNAPII相互作用)。我们将使用体外和体内的方法,包括生化, 功能和基因组分析了解MMEd与RNAPII相互作用的调控及其对细胞周期的影响 转录启动。这些研究将测试拟议的激活机制,该机制将解释 具有相反功能效应的MMEd亚群的意义和检验是否相互转换 MMEd形式之间的差异可以解释MMEd的激活。 在目标2中,我们将确定允许对Mediator-RNAPII进行监管的结构基础 互动。我们推测Tf靶向CKM-MMEd以及随后对MED26和CTD的影响 MMEd结构重排或MMEd中的变化使CTD结合间隙处的相互作用成为可能 构象动力学。对定义明确的中间步骤的结构分析将揭示MMED如何 结构启用激活。我们将使用冷冻-EM来确定各种不同的近原子分辨率图 并使用最先进的图像分析方法来了解它们的构象和 互动动力学。这些研究将揭示支撑主要(最初依赖CTD)的结构性因素 与RNAPII的相互作用)和随后的(RNAPII相互作用和PIC组装的进一步调制)方面 MMEd激活机制的一部分。 在目标3中,我们将研究使MMED激活的MMED中的长程结构重排 通过TF与尾部模块亚基的结合。我们认为,在组成、结构或 通过与TF的相互作用触发的Tail模块的构象动力学可以沿着 MMEd结构到CTD结合间隙,允许MMEd通过 相同的基本激活机制。我们将使用冷冻-EM,图像分析和生物化学来 了解从Tf结合到各种MMEd尾部亚基的信号如何聚合以控制 使用RNAPII的MMED。这一目标中描述的研究将测试所提议的MMED激活的一般性 机制,并揭示了MMEd结构如何集成来自不同目标的各种TF的信号 MMEd亚基。 本申请中提出的研究在概念和技术上都是创新的。我们将测试一部小说 基于“被抑制”和“被抑制”之间相互转换的激活转录启动调控模型 应用多学科方法结合最先进的分子研究MMED的“激活”形式 生物学、基因组学、生物信息学和冷冻-EM/图像分析技术,以了解 哺乳动物PIC的组装和转录启动。我们将结合体外研究,允许检查 建议的机制的各个方面通过体内研究来验证生物学意义和 功能后果。使用最先进的低温电子显微镜和图像分析将使我们能够在之前的基础上 并对错综复杂的MMED的方式达成详细的结构性理解 结构能够激活转录调节。

项目成果

期刊论文数量(13)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Structure and flexibility of nanoscale protein cages designed by symmetric self-assembly.
  • DOI:
    10.1021/ja402277f
  • 发表时间:
    2013-05-22
  • 期刊:
  • 影响因子:
    15
  • 作者:
    Lai, Yen-Ting;Tsai, Kuang-Lei;Sawaya, Michael R.;Asturias, Francisco J.;Yeates, Todd O.
  • 通讯作者:
    Yeates, Todd O.
Interaction of the mediator head module with RNA polymerase II.
  • DOI:
    10.1016/j.str.2012.02.023
  • 发表时间:
    2012-05-09
  • 期刊:
  • 影响因子:
    5.7
  • 作者:
    Cai, Gang;Chaban, Yuriy L.;Imasaki, Tsuyoshi;Kovacs, Julio A.;Calero, Guillermo;Penczek, Pawel A.;Takagi, Yuichiro;Asturias, Francisco J.
  • 通讯作者:
    Asturias, Francisco J.
Iterative stable alignment and clustering of 2D transmission electron microscope images.
  • DOI:
    10.1016/j.str.2011.12.007
  • 发表时间:
    2012-02-08
  • 期刊:
  • 影响因子:
    0
  • 作者:
    Yang Z;Fang J;Chittuluru J;Asturias FJ;Penczek PA
  • 通讯作者:
    Penczek PA
Structure of a RSC-nucleosome complex and insights into chromatin remodeling.
Structure of a designed protein cage that self-assembles into a highly porous cube.
  • DOI:
    10.1038/nchem.2107
  • 发表时间:
    2014-12
  • 期刊:
  • 影响因子:
    21.8
  • 作者:
  • 通讯作者:
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Francisco J Asturias其他文献

Francisco J Asturias的其他文献

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{{ truncateString('Francisco J Asturias', 18)}}的其他基金

Roles of HIV-1 capsid-binding FG-motif containing cellular cofactors in infection
含有细胞辅助因子的 HIV-1 衣壳结合 FG 基序在感染中的作用
  • 批准号:
    10569058
  • 财政年份:
    2022
  • 资助金额:
    $ 48.69万
  • 项目类别:
Ultra-potent HIV capsid inhibitors
超强效 HIV 衣壳抑制剂
  • 批准号:
    10461834
  • 财政年份:
    2020
  • 资助金额:
    $ 48.69万
  • 项目类别:
Ultra-potent HIV capsid inhibitors
超强效 HIV 衣壳抑制剂
  • 批准号:
    10267756
  • 财政年份:
    2020
  • 资助金额:
    $ 48.69万
  • 项目类别:
Ultra-potent HIV capsid inhibitors
超强效 HIV 衣壳抑制剂
  • 批准号:
    10676975
  • 财政年份:
    2020
  • 资助金额:
    $ 48.69万
  • 项目类别:
Ultra-potent HIV capsid inhibitors
超强效 HIV 衣壳抑制剂
  • 批准号:
    10160584
  • 财政年份:
    2020
  • 资助金额:
    $ 48.69万
  • 项目类别:
Structural studies of eukaryotic transcription
真核转录的结构研究
  • 批准号:
    9263971
  • 财政年份:
    2013
  • 资助金额:
    $ 48.69万
  • 项目类别:
Structural studies of eukaryotic transcription
真核转录的结构研究
  • 批准号:
    10004054
  • 财政年份:
    2013
  • 资助金额:
    $ 48.69万
  • 项目类别:
STRUCTURAL STUDIES OF EUKARYOTIC TRANSCRIPTION
真核转录的结构研究
  • 批准号:
    8843003
  • 财政年份:
    2013
  • 资助金额:
    $ 48.69万
  • 项目类别:
STRUCTURAL STUDIES OF EUKARYOTIC TRANSCRIPTION
真核转录的结构研究
  • 批准号:
    8690447
  • 财政年份:
    2013
  • 资助金额:
    $ 48.69万
  • 项目类别:
STRUCTURAL STUDIES OF EUKARYOTIC TRANSCRIPTION
真核转录的结构研究
  • 批准号:
    9057056
  • 财政年份:
    2013
  • 资助金额:
    $ 48.69万
  • 项目类别:

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    23H02481
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    2023
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    $ 48.69万
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    Grant-in-Aid for Scientific Research (B)
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    2320160
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    2023
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    $ 48.69万
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  • 批准号:
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  • 财政年份:
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将细胞骨架力转化为生化信号
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研究人类染色质生化串扰的化学策略
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    $ 48.69万
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    2334134
  • 财政年份:
    2023
  • 资助金额:
    $ 48.69万
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