Evolution and divergence of TBP-mediated transcription activation mechanisms

所有生物体的一个基本过程是遗传信息从DNA到RNA的流动，称为转录，这是由RNA聚合酶介导的。在核被膜包裹的真核生物中，有三种转录不同基因的聚合酶（Pol I， II， III）。塔塔结合蛋白（TBP）是一种转录因子（TF），存在于所有真核生物物种中，是三种RNA pol起始转录所必需的。尽管TBP具有保守性，但其包含的n端结构域在整个进化过程中长度和组成都发生了变化，这暗示了其在真核生物物种转录机制多样化中的潜在作用。我的发现研究项目的长期目标是了解高度保守的转录过程是如何在真核生物中进化和多样化的。为了实现这一目标，我的五年目标是：1)研究TBP的n端结构域在酵母和哺乳动物细胞中的进化作用；2)确定TBP n端结构域的分子作用，因为它与三个RNA pol的转录有关；3)确定脊椎动物特异性tbp相关因子的功能作用。我以前在转录因子方面的工作突出了我在各种技术方面的专业知识，我建议在我的发现研究计划中使用这些技术。二十多年来，科学家们观察到转录的关键调节因子tf在有丝分裂期间被排除在染色体之外，这就提出了有丝分裂后转录如何被重新激活的问题。然而，使用基因编辑、基因组学和活细胞成像，我发现观察到的TF排除是化学交联的产物，这是以前使用的技术的核心。我的发现导致了我们对有丝分裂后转录如何重新启动的理解的范式转变，突出了基因编辑，基因组学和单分子活细胞成像的强大组合，在基础生物学中取得了前所未有的发现。在我的探索研究项目中，我将利用以前的经验和专业知识来研究形成真核生物转录的进化影响。这项研究将有助于培养新兴和跨学科技术的硕士和博士研究生，并支持本科生可以学习和执行独立研究项目的实验室环境。我致力于为不列颠哥伦比亚大学的公平和多样性战略计划中所描述的代表性不足的群体促进公平的培训环境。由于转录是所有生命的核心，因此拟议研究的结果将对自然科学和基因工程各个领域的知识进步产生重大影响。从这项基础生物学研究中获得的知识将对进化和分子生物学领域产生更广泛的影响，并将为未来的基础研究提供信息。

A fundamental process in all living organisms is the flow of genetic information from DNA to RNA, known as transcription, which is mediated by the RNA polymerase enzyme. In eukaryotic organisms where the nucleus is enclosed by a membrane, there are three polymerases (Pol I, II, III) that transcribe different genes. The TATA-binding protein (TBP) which is a transcription factor (TF) found across all eukaryotic species, is required for the initiation of transcription by each of the three RNA Pols. Despite its conserved properties, TBP contains an N-terminal domain that has diversified in length and composition throughout evolution, hinting at a potential role in diversifying transcription mechanisms across eukaryotic species. The long-term goal of my Discovery research program is to understand how the highly conserved transcription process has evolved and diversified across Eukarya. Towards this goal, my five-year objectives are to: 1) examine the evolutionary role of the N-terminal domain of TBP in yeast and mammalian cells; 2) establish the molecular role of TBP N-terminal domain as it relates to transcription by the three RNA Pols; and 3) Determine the functional roles of vertebrate-specific TBP-related factors. My previous work on transcription factors highlights my expertise in the diverse technologies that I propose to use in my Discovery research program. For over two decades, scientists observed that TFs, key regulators of transcription, become excluded from chromosomes during mitosis, raising the question of how transcription is reactivated after mitosis. However, using gene editing, genomics, and live-cell imaging, I discovered that the observed TF exclusion was an artifact of chemical crosslinking, which was central to previously used techniques. My discovery led to a paradigm shift in our understanding of how transcription is restarted after mitosis, highlighting the powerful combination of gene editing, genomics, and single-molecule live-cell imaging to make unprecedented discoveries in basic biology. For my Discovery research program, I will use this previous experience and expertise to study the evolutionary influences that have shaped eukaryotic transcription. This research will help train Masters and PhD graduate students in emerging and interdisciplinary technologies, and support a lab environment where undergraduates can learn and perform independent study projects. I am committed to promoting an equitable training environment for underrepresented groups as described in the University of British Columbia's Equity and Diversity Strategic Plan. Because transcription is central to all life, the results of the proposed studies will have a major impact on advancing knowledge in diverse areas of natural sciences and genetic engineering. Knowledge gained from this basic biological research will have broader implications in the field of evolution and molecular biology, and will inform future basic research.