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

项目摘要/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。为 结构化RNA，高局部稳定性通过逐渐形成模块增强RNA折叠的能力， 但它也增加了失败的几率和后果。对于涉及RNA或 DNA、蛋白质酶是瞬间破坏核酸结构所必需的。此框架提供了 我的团队研究的首要主题。在RNA折叠方面，我们使用的是源自 来测试RNA折叠动力学是否可以被理解并最终预测为 了解作为这些折叠基础的模块化三级触点和结的属性 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 包括三个项目。(1)死盒解旋酶在整个生物学中起着操纵RNA结构的作用。 在过去的12年里，我们使用生化和生物物理方法来描述死箱是如何 解旋酶使用局部RNA解旋来促进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。拟议中的工作 进一步深入研究解旋酶核心的构象变化如何产生依赖于ATP的局部RNA 解旋，这是一个仍然知之甚少的过程，是死盒螺旋酶的一般要求。 我们还将探索Mss 116的生物相互作用和合作伙伴，Mss 116是一种酿酒酵母蛋白，其功能是 线粒体中的一种普通的RNA伴侣。(2)Deah-box家族解旋酶DHX36是一种必需的蛋白质 它与RNA和DNA G-四链结构(G4S)特异性结合，并使用ATP来展开它们。我们的 最近发表的工作确定了G4中断机制的关键要素。拟议中的工作 解决了一个关键问题--DHX36的N-末端结构域如何与G4S特异结合 并验证了DHX36作为端粒酶RNA中G4S的伴侣的假设。 (3)CRISPR-Cas核酸酶在基因编辑等方面具有巨大的应用潜力，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。 各阶段。我们最近应用定量动力学方法来研究Cas12a核酸酶是如何 以实现比基准CAS9更高的靶DNA特异性。我们提出的工作建立在此基础上 通过探索高特异性的物理起源和蛋白质重排是如何联系的来理解 目标是DNA识别。此外，我们将剖析反应步骤和特异性决定因素的前 CrRNA与Cas12a的组装，这还没有被系统地探索。在每个研究领域，我们都在努力 回答可能会给出重要和普遍的见解的基础研究问题。我们的工作也 对理解和治疗疾病有影响，因为这些蛋白质的缺陷与许多 包括癌症和CRISPR-Cas酶在内的疾病已经成为对抗遗传病的关键工具。

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.