Kinetic Dissection of RNA Folding and Proteins that Remodel RNAs and DNAs

RNA 折叠和重塑 RNA 和 DNA 的蛋白质的动力学剖析

• 批准号: 10392905
• 负责人: Rick Russell
• 金额: $ 37.48万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392905
• 关键词: Address; Area; Basic Science; Benchmarking; Binding; Binding Proteins; Biochemical; Biological; Biology; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Structure; Defect; Disease; Dissection; Elements; Enzymes; Family; G-Quartets; Genes; Genetic Diseases; Human; Instruction; Introns; Kinetics; Knowledge; Lead; Life; Link; Malignant Neoplasms; Mediating; Mitochondria; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; N-terminal; Process; Property; Proteins; Publishing; RNA; RNA Folding; Reaction; Research; Saccharomyces cerevisiae; Specificity; Structure; Telomerase RNA Component; Testing; Work; biophysical techniques; combat; helicase; improved; insight; interest; link protein; nuclease; nucleic acid structure; protein function; tool

PROJECT SUMMARY/ABSTRACT The high stability of local structure for RNA and DNA has profound and widespread impacts on life. For structured RNAs, high local stability enhances the ability of RNAs to fold by progressive formation of modules, but it also increases the odds and the consequences of misfolding. For cellular processes involving RNA or DNA, protein enzymes are required to transiently disrupt nucleic acid structure. This framework provides the overarching theme of the research of my group. In the area of RNA folding, we are using model RNAs derived from a group I intron to test whether RNA folding kinetics can be understood and ultimately predicted by understanding the properties of the modular tertiary contacts and junctions that underlie the folding of these RNAs. In the area of protein-mediated changes in nucleic acid structure, our current research interests encompass three projects. (1) DEAD-box helicases function throughout biology to manipulate RNA structures. Over the past 12 years, we have used biochemical and biophysical approaches to delineate how DEAD-box helicases use local RNA unwinding to promote folding and structural rearrangements of RNAs with secondary and tertiary structure, and how a helicase can function as a general RNA chaperone. The proposed work delves further into how conformational changes in the helicase core produce ATP-dependent local RNA unwinding, a process that remains poorly understood and is a general requirement for DEAD-box helicases. We will also explore biological interactions and partners of Mss116, a S. cerevisiae protein that functions as a general RNA chaperone in mitochondria. (2) The DEAH-box family helicase DHX36 is an essential protein that binds specifically to RNA and DNA G-quadruplex structures (G4s) and uses ATP to unfold them. Our recent published work has defined key elements of the mechanism of G4 disruption. The proposed work addresses a key question –how does the N-terminal domain of DHX36, which binds specifically to G4s, assist in their disruption– and tests the hypothesis that DHX36 functions as a chaperone for G4s in telomerase RNA. (3) CRISPR-Cas nucleases have enormous potential for gene editing applications and beyond, but our knowledge of the molecular steps of RNA assembly, DNA targeting, and DNA cleavage are at the very early stages. We recently applied quantitative kinetics approaches to investigate how the Cas12a nuclease is able to achieve higher specificity for target DNA than the benchmark Cas9. Our proposed work builds on this understanding by probing the physical origin of the high specificity and how protein rearrangements are linked to target DNA recognition. In addition, we will dissect the reaction steps and specificity determinants for pre- crRNA assembly with Cas12a, which have not been explored systematically. In each research area, we strive to answer basic research questions that are likely to give important and generalizable insights. Our work also has implications for understanding and treating diseases, as defects in these proteins are linked to many diseases including cancer, and CRISPR-Cas enzymes have emerged as key tools to combat genetic diseases.

项目概要/摘要 RNA和DNA局部结构的高度稳定性对生命有着深远而广泛的影响。 结构化RNA，高局部稳定性增强了RNA通过逐步形成模块而折叠的能力， 但它也会增加涉及 RNA 或 RNA 的细胞过程的错误折叠的可能性和后果。 DNA、蛋白质酶是短暂破坏核酸结构所必需的。 我小组研究的首要主题是在RNA折叠领域，我们使用RNA衍生的模型。 从 I 组内含子中测试 RNA 折叠动力学是否可以被理解并最终预测 了解模块化三级触点和连接点的特性，这些触点和连接点是这些触点折叠的基础 在蛋白质介导的核酸结构变化领域，我们目前的研究兴趣 (1) DEAD-box 解旋酶在整个生物学中发挥着操纵 RNA 结构的作用。 在过去的 12 年里，我们使用生物化学和生物物理方法来描述 DEAD-box 如何 解旋酶利用局部 RNA 解旋来促进具有二级结构的 RNA 的折叠和结构重排 和三级结构，以及解旋酶如何作为通用 RNA 伴侣发挥作用。 进一步深入研究解旋酶核心的构象变化如何产生 ATP 依赖性局部 RNA 解旋，这个过程仍然知之甚少，但它是死盒解旋酶的一般要求。 我们还将探索 Mss116 的生物相互作用和伙伴，Mss116 是一种酿酒酵母蛋白，其功能为 线粒体中的通用 RNA 伴侣 (2) DEAH-box 家族解旋酶 DHX36 是一种必需蛋白质。 特异性结合 RNA 和 DNA G 四链体结构 (G4) 并使用 ATP 来展开它们。 最近发表的工作定义了 G4 破坏机制的关键要素。 解决了一个关键问题——与 G4 特异性结合的 DHX36 N 端结构域如何协助 并测试了 DHX36 作为端粒酶 RNA 中 G4 的伴侣的假设。 (3) CRISPR-Cas 核酸酶在基因编辑应用及其他领域具有巨大的潜力，但我们的 对 RNA 组装、DNA 靶向和 DNA 切割的分子步骤的了解还处于非常早期的阶段 我们最近应用定量动力学方法来研究 Cas12a 核酸酶如何发挥作用。 以实现比基准 Cas9 更高的目标 DNA 特异性。 通过探究高特异性的物理起源以及蛋白质重排如何联系来理解 此外，我们将剖析预-DNA 识别的反应步骤和特异性决定因素。 crRNA与Cas12a的组装，尚未被系统地探索过，在每个研究领域，我们都在努力。 回答可能为我们的工作提供重要且普遍见解的基础研究问题。 对理解和治疗疾病具有重要意义，因为这些蛋白质的缺陷与许多疾病有关 包括癌症在内的疾病和 CRISPR-Cas 酶已成为对抗遗传疾病的关键工具。