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.

项目总结/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。为 结构化RNA，高局部稳定性增强RNA通过逐步形成模块折叠的能力， 但这也增加了错误折叠的可能性和后果。对于涉及RNA或 需要DNA、蛋白质酶来瞬时破坏核酸结构。该框架提供了 我的团队研究的首要主题。在RNA折叠领域，我们使用的是来自 以测试RNA折叠动力学是否可以理解，并最终预测， 理解模块化第三级接触和连接的特性，这些接触和连接是这些折叠的基础。 RNA。在蛋白质介导的核酸结构变化领域，我们目前的研究兴趣是 包括三个项目。(1)DEAD盒解旋酶在整个生物学中发挥作用，以操纵RNA结构。 在过去的12年里，我们已经使用生物化学和生物物理学的方法来描述死亡盒是如何被激活的。 解旋酶使用局部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一般的RNA伴侣发挥作用。拟议工作 深入研究了解旋酶核心的构象变化如何产生依赖ATP的局部RNA 解旋，一个仍然知之甚少的过程，是DEAD盒解旋酶的一般要求。 我们还将探讨生物相互作用和合作伙伴的Mss 116，一个S。酿酒蛋白，其功能为 线粒体中的一种普通RNA伴侣。(2)DEAH盒家族解旋酶DHX 36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将其展开。我们 最近发表的工作已经定义了G4破坏机制的关键要素。拟议工作 解决了一个关键问题-DHX 36的N-末端结构域如何与G4特异性结合， 并测试了DHX 36作为端粒酶RNA中G4分子伴侣的假设。 (3)CRISPR-Cas核酸酶在基因编辑应用及其他方面具有巨大的潜力，但我们的研究表明， 对RNA组装、DNA靶向和DNA切割的分子步骤的了解还处于早期阶段 阶段我们最近应用定量动力学方法来研究Cas 12 a核酸酶如何能够 以实现比基准Cas9对靶DNA更高的特异性。我们建议的工作建立在此基础上 通过探测高特异性的物理起源以及蛋白质重排如何联系起来来理解 以DNA识别为目标此外，我们将剖析预处理的反应步骤和特异性决定因素， crRNA与Cas 12 a的组装，这尚未被系统地探索。在每一个研究领域，我们努力 回答可能提供重要和可推广见解的基础研究问题。我们的工作还 这些蛋白质的缺陷与许多疾病有关， 包括癌症在内的遗传性疾病，CRISPR-Cas酶已经成为对抗遗传性疾病的关键工具。