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.

项目摘要：RNA聚合酶（RNAP）的运行依赖于许多构象变化。 在真核转录期间，RNA聚合酶II（POL II）在其DNA模板中遇到氧化病变 通常会导致失误和转录失速。这些事件导致皮肤肿瘤的生长 癌症。结核分枝杆菌（MTB）引起致命的结核病，并导致100万人死亡 每年。 MTB RNAP的转录起始复合物，尤其是DNA载荷是有效的靶标 用于开发抗生素。揭示转录计划的动力学可以提供新颖 对原核转录的机械洞察力，并极大地支持对抑制的理解 针对MTB RNAP的抗生素机制。这两个转录驱动器中的两个重要生物学问题 我们使用广义主方程（GME）开发新方法，以建模生物分子 构象变化。我的小组已经成功地开发了明确考虑的GME方法 生物分子动力学的内存功能和优于流行的马尔可夫状态模型（MSM）方法。 但是，作为一种新兴方法，当前GME的实施在估算时容易出现不稳定 复杂RNAP系统的内存功能。我们在这里提出新方法来构建GME模型。我们的 具体目的是：1。开发新的GME方法来建模组成更改。具体而言，推导 解决GME的新理论（IGME），以开发GME的有效实现以增强数值 从分子动力学（MD）仿真轨迹计算内存内核时的稳定性，并创建一个 量身定制的用于构建GME模型的协议，以研究生物分子构象变化。我们的初步 工作表明，提出的IGME方法极大地超过了GME的原始实现 对生物分子动力学的强大而准确的预测，尤其是对于复杂的RNAP系统。 2 揭示几种关键构象变化的动态耦合（即，NTP的加载，旋转 DNA损坏的DNA碱基和DNA模板上的POL II的易位）导致转录 诱变和/或失速。具体而言，构建GME模型以阐明8-氧的分子机制 鸟嘌呤（8OG）和鸟尼二羟富卫生（GH）病变诱导ATP失调和/或转录 存储。 3。阐明转录倡议的分子机制及其对MTB RNAP的抑制。 具体而言，要构建GME模型以揭示MTB RNAP加载门无DNA的动力学，并且 进一步揭示从部分形成的转录气泡过渡到完全形成的动力学 泡泡是一种构象变化，涉及MTB RNAP的大门开口和DNA放松。我们进一步瞄准 了解多种抗生素化合物的识别机制，包括粘霉素（Myx）和 靶向加载门运动的fidaxomicin（FDX），sorangicin（SOR）抑制完整的形成 转录气泡。这些机械洞察力将有助于与药物作斗争的新抑制剂的合理设计 从长远来看MTB的电阻。在整个研究中，我们将与实验紧密合作 进行生化，时间分辨X射线和冷冻EM实验的合作者，以测试和验证我们 预测。我们创新的GME方法将提供一个通用的计算框架来建模功能 生物分子的构象变化。我们开发的协议和相关的代码开发 MSMBuilder软件将广泛受益于生物物理学社区。