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: 9908117
• 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/9908117
• 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; Structural defect; 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.

项目摘要/摘要 RNA和DNA的局部结构的高稳定性对生命产生了深远而宽度的影响。为了 结构化的RNA，高局部稳定性可以增强RNA通过模块的逐步形成而折叠的能力， 但这也增加了错误折叠的几率和后果。用于涉及RNA或的细胞过程 DNA，蛋白质酶需要瞬时破坏核酸结构。这个框架提供了 我小组研究的总体主题。在RNA折叠的区域，我们使用的是衍生的模型RNA 从I组内含子到测试RNA折叠动力学是否可以理解并最终通过 了解这些折叠的基础的模块化三级触点和连接的特性 RNA。在蛋白质介导的核酸结构变化的领域，我们目前的研究兴趣 包括三个项目。 （1）死盒解旋酶在整个生物学过程中发挥作用，以操纵RNA结构。 在过去的12年中，我们使用生化和生物物理方法来描绘死箱如何 解旋酶使用局部RNA放松，以促进RNA的折叠和结构重排 和三级结构，以及解旋酶如何充当一般的RNA伴侣。拟议的工作 进一步研究旋转酶核心的会议变化如何产生依赖ATP的局部RNA 放松身心，这一过程仍然很糟糕，并且是对死箱解旋酶的一般要求。 我们还将探索MSS116的生物学相互作用和合作伙伴，这是一种酿酒酵母蛋白，起作用为 线粒体中的一般RNA链酮。 （2）DEAH-box家族解旋酶DHX36是必不可少的蛋白质 这与RNA和DNA G四链体结构（G4S）特别结合，并使用ATP展开它们。我们的 最近发表的工作定义了G4破坏机制的关键要素。拟议的工作 解决一个关键问题 - DHX36的N末端域如何专门绑定到G4S，协助 在它们的破坏中 - 检验了DHX36在端粒酶RNA中充当G4S的伴侣的假设。 （3）CRISPR-CAS核具有巨大的基因编辑应用的潜力，但是我们的 了解RNA组件的分子步骤，DNA靶向和DNA裂解很早就 阶段。我们最近采用定量动力学方法来研究Cas12a核酸酶如何能够 比基准Cas9获得目标DNA的特异性更高。我们提出的工作以此为基础 通过探测高特异性的物理起源以及如何联系蛋白质重排 靶向DNA识别。此外，我们将剖析反应步骤和特异性确定剂的前 用CAS12A组装的CRRNA组件，尚未系统地探索。在每个研究领域，我们努力 回答可能提供重要且可推广的见解的基础研究问题。我们的工作也是如此 对理解和治疗疾病具有影响，因为这些蛋白质中的缺陷与许多 包括癌症和CRISPR-CAS酶在内的疾病已成为对抗遗传疾病的关键工具。