Theory and Modeling of Functional Conformational Changes of RNA Polymerases

RNA聚合酶功能构象变化的理论和建模

• 批准号: 10656962
• 负责人: Xuhui Huang
• 金额: $ 35.83万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656962
• 关键词: Address; Adverse effects; Algorithms; Antibiotics; Biochemical; Biological; Biophysics; Cessation of life; Communities; Complex; Computer software; Computing Methodologies; Coupling; Cryoelectron Microscopy; DNA; DNA Damage; DNA lesion; DNA-Directed RNA Polymerase; Development; Drug resistant Mycobacteria Tuberculosis; Equation; Event; Foundations; Gene Expression; Genetic Transcription; Knowledge; Length; Lesion; Memory; Methodology; Methods; Modeling; Molecular; Molecular Conformation; Motion; Mutagenesis; Mycobacterium tuberculosis; Mycobacterium tuberculosis complex; Outcome; Polymerase; Protocols documentation; RNA Polymerase II; Roentgen Rays; Role; Rotation; Route; Scheme; Series; Skin Cancer; System; Testing; Thermus; Time; Transcription Initiation; Tuberculosis; Work; base; code development; computer framework; discrete time; experimental study; fighting; flexibility; guanidinohydantoin; inhibitor; innovation; insight; molecular dynamics; novel; operation; rational design; theories; time interval; tumor growth

Project Summary: The operation of RNA polymerases (RNAPs) relies on numerous conformational changes. During eukaryotic transcription, RNA Polymerase II (Pol II) encountering oxidative lesions in its DNA template often leads to misincorporation and transcriptional stalling. These events contribute to tumor growth in skin cancer. Mycobacterium tuberculosis (Mtb) causes lethal tuberculosis and is responsible for over 1 million deaths per year. Transcription initiation complexes of Mtb RNAP, especially the DNA loading gate, are effective targets for the development of antibiotics. Revealing the dynamics of transcription initiation can thus provide novel mechanistic insights into prokaryotic transcription and greatly facilitate the understanding of inhibition mechanisms for antibiotics targeting Mtb RNAP. These two important biological problems in transcription drive us to develop novel methodology using the generalized master equation (GME) to model biomolecular conformational changes. My group has been successful in developing GME methods that explicitly consider the memory functions of biomolecular dynamics and outperform the popular Markov State Model (MSM) method. However, as an emerging approach, the current implementation of GME is prone to instability when estimating memory functions for complex RNAP systems. We here propose novel methods to build GME models. Our specific aims are: 1. To develop new GME methods to model conformational changes. Specifically, to derive a new theory (IGME) to solve the GME, to develop efficient implementations of the GME to enhance numerical stability when computing memory kernels from molecular dynamics (MD) simulation trajectories, and to create a protocol tailor-made for building GME models to study biomolecular conformational changes. Our preliminary work shows that the proposed IGME method greatly outperforms the original implementation of GME in yielding robust and accurate predictions of the biomolecular dynamics, especially for the complex RNAP system. 2. To reveal how the dynamic coupling of several key conformational changes (i.e., the loading of NTP, the rotation of the damaged DNA base, and the translocation of Pol II on the DNA template) leads to transcriptional mutagenesis and/or stalling. Specifically, to construct GME models to elucidate molecular mechanisms of 8-oxo- guanine (8OG) and Guanidinohydantoin (Gh) lesions induced ATP misincorporation and/or transcriptional stalling. 3. To elucidate the molecular mechanisms of transcriptional initiation and its inhibition of Mtb RNAP. Specifically, to construct GME models to reveal the dynamics of the Mtb RNAP’s loading gate without DNA, and to further reveal the dynamics for the transition from a partially formed transcription bubble to a fully formed bubble, a conformational change involving both Mtb RNAP’s gate opening and DNA unwinding. We further aim to understand the recognition mechanisms of multiple antibiotic compounds, including Myxopyronin (Myx) and Fidaxomicin (Fdx) that target the loading gate motion, and Sorangicin (Sor) that inhibits the formation of the full transcription bubble. These mechanistic insights will facilitate the rational design of new inhibitors fighting drug resistance of Mtb in the long term. Throughout our studies, we will work closely with our experimental collaborators to conduct biochemical, time-resolved X-ray, and Cryo-EM experiments to test and validate our predictions. Our innovative GME methods will provide a general computational framework to model functional conformational changes of biomolecules. Our developed protocol and associated code development in the MSMBuilder software will widely benefit the biophysics community.

项目概述：RNA聚合酶（RNAP）的运作依赖于许多构象变化。 在真核生物转录过程中，RNA聚合酶II（Pol II）在其DNA模板中遇到氧化损伤 常常导致错误掺入和转录停滞。这些事件有助于皮肤中的肿瘤生长 癌结核分枝杆菌（Mtb）导致致命的结核病，并造成100多万人死亡 每一年。Mtb RNAP的转录起始复合物，特别是DNA加载门，是有效的靶点 抗生素的发展。因此，揭示转录起始的动力学可以提供新的 对原核生物转录机制的深入了解，极大地促进了对抑制作用的理解。 针对Mtb RNAP的抗生素的机制。转录驱动中的两个重要生物学问题 我们开发了一种新的方法，使用广义主方程（GME）来模拟生物分子 构象变化我的小组已经成功地开发了明确考虑 生物分子动力学的记忆功能，并优于流行的马尔可夫状态模型（MSM）方法。 然而，作为一种新兴的方法，GME的当前实施在估计时容易出现不稳定性 复杂RNAP系统的存储功能。在这里，我们提出了新的方法来建立GME模型。我们 具体目标是：1.开发新的GME方法来模拟构象变化。具体地说，为了得到一个 新理论（IGME）来解决GME，开发GME的有效实现，以增强数值计算能力。 当从分子动力学（MD）模拟轨迹计算存储器内核时， 为构建GME模型以研究生物分子构象变化而量身定制的方案。我们的初步 工作表明，所提出的IGME方法大大优于原来的实现GME在产量 生物分子动力学的鲁棒和准确的预测，特别是对于复杂的RNAP系统。2.到 揭示了几个关键构象变化的动态耦合（即，NTP的加载， 损伤的DNA碱基，以及DNA模板上Pol II的易位）导致转录 诱变和/或停滞。具体而言，构建GME模型以阐明8-氧代- 鸟嘌呤（8 OG）和胍基海因（Gh）损伤诱导ATP错误掺入和/或转录 拖延时间3.阐明结核分枝杆菌RNAP转录起始及其抑制的分子机制。 具体地，构建GME模型以揭示没有DNA的Mtb RNAP的加载门的动力学，以及 为了进一步揭示从部分形成的转录泡到完全形成的转录泡的转变的动力学， 气泡，涉及Mtb RNAP的门打开和DNA解旋的构象变化。我们进一步致力于 了解多种抗生素化合物的识别机制，包括Myxopyronin（Myx）和 靶向加载门运动的非达霉素（Fdx）和抑制完整的细胞周期的形成的Sorangicin（Sor）。 转录气泡。这些机制的见解将有助于合理设计新的抑制剂， 结核分枝杆菌的长期耐药性。在我们的研究中，我们将与我们的实验密切合作， 合作者进行生化，时间分辨X射线和冷冻EM实验，以测试和验证我们的 预测。我们创新的GME方法将提供一个通用的计算框架， 生物分子的构象变化。我们开发的协议和相关的代码开发在 MSMBuilder软件将使生物物理学界广泛受益。