Theory and Modeling of Functional Conformational Changes of RNA Polymerases

项目摘要：RNA聚合酶(RNAP)的运行依赖于大量的构象变化。 在真核转录过程中，RNA聚合酶II(POL II)的DNA模板遇到氧化损伤 经常导致错误整合和转录停滞。这些事件促进了皮肤肿瘤的生长。 癌症。结核分枝杆菌(Mtb)会导致致命的结核病，并导致100多万人死亡。 每年。Mtb RNAP的转录起始复合体，特别是dna载入门，是有效的靶点。 用于抗生素的开发。因此，揭示转录启动的动力学可以提供新的 对原核生物转录的机械洞察，并极大地促进了对抑制的理解 针对结核分枝杆菌RNAP的抗生素机制。转录驱动中的这两个重要的生物学问题 美国将开发使用广义主方程(GME)模拟生物分子的新方法 构象变化。我的团队已经成功地开发了GME方法，这些方法明确地考虑了 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，目前实施的GME在估计时容易出现不稳定的情况 复杂RNAP系统的内存功能。我们在这里提出了建立GME模型的新方法。我们的 具体目标是：1.发展新的GME方法来模拟构象变化。具体地说，要派生一个 新理论(IGME)求解GME，发展高效的GME实现增强数值 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 为建立GME模型以研究生物分子构象变化而量身定做的协议。我们的预赛 工作表明，所提出的IGME方法在成品率方面明显优于GME的原始实现 稳健和准确的生物分子动力学预测，特别是对于复杂的RNAP系统。2.至 揭示了几个关键构象的动态耦合是如何变化的(即NTP的负载、 DNA碱基受损和DNA模板上POL II的移位)导致转录 诱变和/或拖延。具体地说，构建GME模型以阐明8-氧代-2-甲氧基-4-甲氧基苯乙酮的分子机理。 鸟嘌呤(80G)和海因(Gh)损伤引起的ATP错掺入和/或转录 在拖延时间。3.阐明Mtb RNAP转录启动及其抑制的分子机制。 具体地说，构建GME模型以揭示Mtb RNAP在没有DNA的情况下装载门的动力学，以及 为了进一步揭示从部分形成的转录泡泡到完全形成的转录泡泡的转变动力学 泡泡，一种构象变化，涉及Mtb RNAP的门打开和DNA解离。我们进一步的目标是 了解多种抗生素化合物的识别机制，包括粘比罗宁(Myx)和 靶向加载门运动的非达克索米星(FDX)和抑制充盈的形成的索兰金(SOR) 转录泡泡。这些机理上的见解将有助于合理设计新的抗药物抑制剂 结核分枝杆菌的长期耐药性。在我们的整个研究过程中，我们将与我们的实验 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来模拟泛函 生物分子的构象变化。我们开发的协议和相关代码开发在 MSMBuilder软件将使生物物理界广泛受益。

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.