DNA/RNA聚合酶“聚合-剪切校验”机制的统计物理学研究

DNA/RNA polymerases (DNAP/RNAP) are the most important molecular machines in cell. They copy the genetic information from the DNA template with extremely high fidelity, by catalyzing the copolymerization of the nascent DNA or RNA according to the template. Experiments have revealed that both machines achieve high fidelity through the cooperation between the polymerase pathway and proofreading pathway. Kinetic and thermodynamic studies on the cooperation mechanism are crucial to understand the design principle of DNAP/RNAP. Unfortunately, kinetic models and related investigation on non-equilibrium thermodynamics are still lacking. Even worse, the higher-order terminal effects which is crucial for real DNAP/RNAP to achieve high fidelity are rarely mentioned in existing models. Hence, in this project, we will first build appropriate polymerase-and-proofreading models to incorporate the higher-order effects, and then conduct comprehensive studies on the kinetics and thermodynamics of these model, with a focus to uncover the subtile trade-off between the fidelity, the overall reaction rate and the overall energy dissipation. In addition, since RNAP often works in group, we will also investigate the collective behavior of multi-RNAP systems and try to address how the cooperation between motors changes the overall rate and overall fidelity of the whole system. Another important fact which is not considered in all the existing models is the template-sequence effect, i.e., the base sequence of the template can put a significant impact on the kinetics and fidelity of DNAP/RNAP. We will also try to study the sequence-related kinetics and thermodynamics in this project.

DNA/RNA聚合酶（DNAP、RNAP）是细胞中最重要的分子机器，它们均以DNA为模板催化新链共聚合成，以超高保真度拷贝遗传信息。实验发现，它们都通过“聚合-剪切校验”两通路协作来获得高保真度。从动力学、能量学两方面研究这一协作机制，对深入理解其设计原理极其重要。然而，相关共聚动力学建模、非平衡热力学分析等工作至今仍很匮乏，尤其是对保真度至关重要的高阶末端效应，现有模型几乎没有涉及。为此，本项目将基于结构、生化数据，构建包含高阶效应的 “聚合-剪切”模型，通过模拟与解析计算，系统研究DNAP、RNAP动力学及热力学，特别要阐明保真度与聚合总速度、熵产生之间的关系。对于RNAP多马达系统，我们将深入研究其协作行为对总体转录速度和保真度的影响。此外，模板碱基序列对DNA复制和转录的进程和保真度都有显著影响，而这一点在现有模型中都未能体现，本项目也将研究序列相关的动力学和热力学问题。

DNA聚合酶（DNAP）、RNA聚合酶（RNAP）是遗传信息复制、转录的核心分子机器。它们是如何做到高保真地传递遗传信息的？对这一问题的理论研究已有40多年的历史，主流理论是1970年代建立的动态校对学说，已被成功应用于分子生物学的很多方面。但后续研究表明该学说并不适用于DNA复制及RNA转录。结构生物学和生物化学的研究揭示DNAP、RNAP具有独特的保真机制，它们都通过两道关卡来筛除误配对核苷酸，即聚合模式下的初次筛选以及外切模式下的二次校对，这些过程中还存在对保真度其决定性作用的最近邻甚至高阶近邻相互作用，这完全不同于动态校对学说的物理图像。由于这类“聚合-外切校对”过程对应的动力学方程组通常极大甚至无穷大，在解析计算上难以处理，导致相关理论研究最近十年才逐渐开展。在本项目中，我们先后提出了两类能够解析求解上述问题的数学方法。一类是定态共聚分析方法，适用于DNA模板序列效应可忽略的情况，可精确研究其动力学和非平衡热力学问题；另一类是首达过程分析方法，适用于具有模板序列效应的一般情况，可精确研究其长时间动力学性质。这两类方法都能系统、精确地处理“聚合-外切校对”过程中的任意阶近邻效应。采用这些方法，我们首次推导了DNAP、RNAP保真度、速度等关键物理量的近似解析解，为保真度的精确计算提供了坚实的理论基础。基于该结果，我们对DNAP保真度的主流实验分析方法的理论基础提出了质疑并进行了全面剖析，指出了其合理性和局限性，对进一步的精确测量具有重要的参考价值。