Understanding Gene Transcription from First-Principles: A single-molecule study

描述（由申请人提供） 摘要：基因表达的精确调控对于多细胞生物体的正常发育和维持成体组织中的细胞稳态至关重要。基因表达的失调是越来越多的人类疾病的标志，许多药物直接靶向关键基因转录因子。然而，目前的治疗影响转录在一个 泛基因组规模，没有特异性，也没有微调基因活性的能力。我们缺乏 对转录调控的详细途径的理解排除了设计靶向治疗的可能性。此外，目前的实验工具不能解决涉及人mRNA转录的生化系统的复杂性，该系统由多组分核心转录机制和多个额外层的调节因子、共激活因子、阻遏物和染色质重塑复合物组成。关键的是，目前没有实验方法可以观察活细胞内这些因子的动态，剖析转录动力学，确定受调控的限速步骤，并最终确定转录的关键步骤。 揭示机械原理。我的目标是开发能够提供高分辨率，动态数据的光学成像和光谱工具，以便于详细描述转录调控的分子术语。在此期间，我设想朝着这个方向完成以下工作：（1）创造创新技术，使活细胞内单分子水平的复杂生化过程实时可视化成为可能;（2）识别转录因子调节mRNA合成周期中限速步骤的体内机制;（3）为理解基因表达的信号整合和组合控制提供了框架。拟议的研究建立在我在物理和生物科学的边界独特的专业知识，并寻求创造一个新的思想和方法的合成，以新的策略来理解和控制基因表达，在某种程度上相关的医学。 公共卫生相关性：转录是基因表达的第一步，也是最受调控的一步。异常转录与越来越多的疾病相关，并且几种药物直接靶向转录因子。本项目旨在阐明核心分子转录机制的功能，并为控制其调控提供新的治疗应用策略。

DESCRIPTION (Provided by the applicant) Abstract: The accurate regulation of gene expression is crucial for proper development of multi-cellular organisms and for maintaining cell homeostasis in adult tissues. De-regulation of gene expression is the hallmark of an ever-increasing number of human diseases and many drugs directly target key gene transcription factors. Current therapies however affect transcription on a pan-genomic scale, without specificity and without the ability to fine-tune gene activity. Our lack of understanding of the detailed pathways underlying transcriptional regulation precludes designing targeted therapies. Moreover, current experimental tools cannot address the complexity for the biochemical system involved in human mRNA transcription, comprised by multi-component core transcription machinery and multiple additional layers of regulatory factors, co-activators, repressors and chromatin remodeling complexes. Critically, no current experimental method can observe the dynamics of these factors inside a live cell, dissect transcription kinetics, identify rate-limiting steps that are subject to regulation, and ultimately uncover mechanistic principles. My goal is to develop the enabling optical imaging and spectroscopy tools that can provide high-resolution, dynamic data, to facilitate describing transcription regulation in detailed molecular terms. During the period of this award I envision accomplishing the following towards this direction: (1) create innovating technologies that will make possible to visualize complex biochemical processes at the single-molecule level, in real-time, inside living cells; (2) discern in vivo mechanisms by which transcription factors regulate rate-limiting steps in the cycle of mRNA synthesis; (3) provide a framework for understanding signal integration and combinatorial control of gene expression. The proposed research builds upon my unique expertise at the border of physical and biological sciences, and seeks to create a new synthesis of ideas and methodologies towards novel strategies to understand and control gene expression, in a way relevant to medicine. Public Health Relevance: Transcription is the first and most highly regulated step in gene expression. Aberrant transcription is associated with an ever-growing number of diseases and several drugs directly target transcription factors. This project aims at elucidating the function f the core molecular transcription machinery and provide new strategies for controlling its regulation for therapeutic applications.