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

基本信息

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
项目类别：
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

项目摘要

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) 的运行依赖于众多构象变化。在真核转录过程中，RNA 聚合酶 II (Pol II) 的 DNA 模板遭遇氧化损伤这些事件通常会导致错误整合和转录停滞，从而导致皮肤中的肿瘤生长。结核分枝杆菌 (Mtb) 会导致致命的结核病，并导致超过 100 万人死亡。 Mtb RNAP 的转录起始复合物，尤其是 DNA 加载门，是有效的靶点。因此，揭示转录起始的动态可以为抗生素的开发提供新的思路。对原核转录的机制了解并极大地促进对抑制的理解针对 Mtb RNAP 的抗生素机制。转录驱动中的这两个重要的生物学问题。我们开发新的方法，使用广义主方程（GME）来模拟生物分子我的小组已成功开发出明确考虑构象变化的 GME 方法。生物分子动力学的记忆功能，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，目前GME的实现在估计时容易出现不稳定的情况。我们在这里提出了构建 GME 模型的新方法。具体目标是： 1. 开发新的 GME 方法来模拟构象变化。新理论（IGME）来解决 GME，开发 GME 的有效实现，以增强数值从分子动力学 (MD) 模拟轨迹计算内存内核时的稳定性，并创建我们的初步方案是为构建 GME 模型以研究生物分子构象变化而量身定制的。工作表明，所提出的 IGME 方法在产量方面大大优于 GME 的原始实现生物分子动力学的稳健和准确的预测，特别是对于复杂的 RNAP 系统 2. 揭示几个关键构象变化（即 NTP 的加载、受损的 DNA 碱基以及 Pol II 在 DNA 模板上的易位）导致转录具体来说，构建 GME 模型以阐明 8-oxo- 的分子机制。鸟嘌呤 (8OG) 和胍基乙内酰脲 (Gh) 损伤诱导 ATP 错误掺入和/或转录 3. 阐明Mtb RNAP转录起始及其抑制的分子机制。具体来说，构建 GME 模型来揭示 Mtb RNAP 加载门在没有 DNA 的情况下的动态，以及进一步揭示从部分形成的转录气泡到完全形成的转录气泡转变的动力学气泡，涉及 Mtb RNAP 大门打开和 DNA 解旋的构象变化。了解多种抗生素化合物的识别机制，包括粘菌素 (Myx) 和 Fidaxomicin (Fdx) 靶向加载门运动，Sorangicin (Sor) 抑制完整的形成这些机制见解将有助于合理设计新的抗药物抑制剂。从长远来看，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 射线和冷冻电镜实验来测试和验证我们的我们创新的 GME 方法将为函数式建模提供通用计算框架。我们开发的协议和相关代码开发。 MSMBuilder 软件将使生物物理学界广泛受益。