NMR and EPR Studies of Ribozyme Catalytic Mechanisms

描述(由申请人提供)：这个项目的首要目标是对催化RNA分子或核酶的作用机制有一个详细的生物物理理解。核酶衍生物目前正在开发中，作为治疗一些毁灭性疾病的药物。在设计衍生物时，对核酶机制的基本了解是至关重要的，同时在保持所需催化活性的同时，还需要改善药理性质。我们的论文认为，核酶的静态结构不足以理解其作用机制，结构功必须与分子动力学、它与多价金属离子的相互作用以及这些金属离子对分子结构和动力学的影响相结合。我们将应用现代磁共振技术，包括异核核磁共振自旋弛豫和先进的EPR实验，以提供与这些问题直接相关的独特信息，从而将结构研究与生化能量学联系起来。重点介绍核磁共振自旋弛豫研究核酶构象的动力学性质。在表征良好的发夹状核酶中，我们将使用活性和非活性序列变体中的核磁共振自旋弛豫来研究分子的动态性质，并作为三级结构形成(对接)的函数。我们将继续使用集成的EPR和核磁共振方法，来描述多价金属离子对核酶结构和动力学的影响。多价金属离子对催化至关重要，但在这个系统中不直接参与化学。我们还对U6 SnRNA感兴趣，它是真核细胞mRNA剪接装置的催化关键成分。在这个系统中，我们将研究一种经过生物化学鉴定的金属离子的结构，它似乎参与了反应化学，无论是在形成其直接结合部位的内部茎环中，还是在更大的复合体中，包括前mRNA序列。对这两个系统的研究将为构象动力学、金属离子辅助因子连接和RNA催化功能之间的相互关系提供互补和协同的视角。简而言之，我们提出了一个关于技术进步和新应用的综合生物物理计划，这将极大地推动核酶领域向分子水平了解这些迷人的催化剂。

DESCRIPTION (provided by applicant): The overarching goal of this project is to develop a detailed biophysical understanding of the mechanism of action of catalytic RNA molecules, or ribozymes. Ribozyme derivatives are currently under development as therapeutics for a number of devastating diseases. A fundamental understanding of ribozyme mechanism is critical in the design of derivatives, with necessary improvements in pharmacological properties that simultaneously maintain the desired catalytic activity. Our thesis, is that, the static structure of a ribozyme is insufficient to understand its mechanism of action, and that structural work must be coupled with studies of the molecule's dynamics, its interactions with multivalent metal ions, and the effects of those metal ions on molecular structure and dynamics. We will apply contemporary magnetic resonance techniques, including heteronuclear NMR spin relaxation and advanced EPR experiments, to provide unique information that bears directly on these issues and, thus, to link structural studies with biochemical energetics. The dynamic properties of ribozyme conformations, as studied by NMR spin relaxation, will be emphasized. In the well characterized hairpin ribozyme, we will investigate the dynamic properties of the molecule using NMR spin relaxation in active vs. inactive sequence variants, and as a function of tertiary structure formation (docking). We will proceed to use an integrated EPR and NMR approach, to delineate the effects of multivalent metal ions, which are crucial for catalysis, but do not participate directly in chemistry in this system, on the ribozyme's structure and dynamics. We are also interested in the U6 snRNA, a catalytically critical component of the eukaryotic mRNA splicing apparatus. In this system, we will investigate the structure of a biochemically-identified metal ion, which appears to participate in reaction chemistry, both in the internal stemloop that forms its immediate binding site, and in larger complexes, including pre-mRNA sequences. Studies of these two systems will provide complementary and synergistic perspectives on the interrelationships among conformational dynamics, metal ion cofactor ligation, and catalytic function in RNA. In short, we propose an integrated biophysical program of technological progress and novel applications, that will significantly advance the ribozyme field toward a molecular-level understanding of these fascinating catalysts.