Dissecting functional cooperation among subunits in a catalytic ribonucleoprotein

剖析催化核糖核蛋白亚基之间的功能合作

基本信息

批准号：
9750734
负责人：
Venkat Gopalan
金额：
$ 43.56万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-26 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9750734
关键词：
Acylation Address Affect Affinity Archaea Binding Binding Proteins Binding Sites Biogenesis Biological Assay Biology Catalysis Catalytic RNA Complex Consensus Coupled Coupling Disease Dissociation Electrons Enzymes Exhibits Fluorescence Fluorescence Resonance Energy Transfer Genetic Genetic Diseases Glycine decarboxylase Goals Guide RNA Holoenzymes Human Hydroxyl Radical In Vitro Kinetics Lead Life Link Maps Mass Spectrum Analysis Mediating Messenger RNA Methods Mission Modeling Molecular Molecular Conformation Nerve Degeneration Nucleotides Play Primer Extension Protein Subunits Proteins Proxy Public Health RNA RNA Conformation RNA Probes RNA Splicing RNA-Protein Interaction RNase P Resolution Ribonucleoproteins Ribosomes Roentgen Rays Role Route Sampling Signal Recognition Particle Site Structure Structure-Activity Relationship Surface Surveys Testing Tissues Transfer RNA Translations Untranslated RNA Variant Work base driving force enzyme substrate complex experimental study functional outcomes human disease in vivo innovation insight ion mobility mutant nuclease reconstitution single molecule tRNA Precursor trait

项目摘要

SUMMARY Our scientific objective is to understand how proteins modulate the function of ribonucleoprotein (RNP) enzymes through structural changes to their associated catalytic RNA. This goal is highly relevant to public health due to the growing appreciation for the roles of RNPs in tissue complexity and human diseases. In this proposal, we will use RNase P as a model to test our postulate that the versatility of RNPs is due to protein- mediated structural changes in their RNA cores. Although the primary function of RNase P is 5ʹ′-maturation of precursor tRNAs, recent findings suggest an expanded functional mission that includes biogenesis of eukaryotic non-coding RNAs. Eukaryotic and archaeal RNase P consist of a catalytic RPR (RNase P RNA) and multiple (4-10) RPPs (RNase P Proteins), unlike the simpler bacterial version (1 RPR + 1 RPP). Because all RPRs are active on their own in vitro, the need for multiple archaeal and eukaryotic RPPs is unclear. We found from step-wise reconstitutions of archaeal RNase P that its assembly intermediates comprising partial suites of five RPPs and the RPR exhibit activity and fidelity of processing in between the RPR alone or the full holo- enzyme (RPR + all RPPs). These findings motivate our central hypothesis that binding of RPPs to specific RPR regions independently and collectively mediates RNA structural changes essential for assembly and catalysis. We will address this hypothesis with two specific aims to delineate structure-function relationships of intermediates en route to assembly of the full RNP: (1) Dissect the structural basis for the distinct roles of archaeal RPPs in aiding RPR catalysis, and (2) map the assembly landscape of archaeal RPPs on the RPR. To study how RPPs guide the RPR towards its functional state, we propose an innovative combination of site- specific and global structural methods coupled to direct functional readouts. In Aim 1, we will probe archaeal RPR structural changes induced by different suites of RPPs at nucleotide resolution using SHAPE-Seq (selective 2ʹ′-hydroxyl acylation analyzed by primer extension sequencing), a high throughput method to probe RNA structures. Inferences from SHAPE-Seq, linking structural changes to functional outcomes, will be guided by the RNA-protein contact sites obtained from tethered-nuclease mapping and validated using assays of RPR mutants. In Aim 2, we will survey the hierarchy and cooperation during RNase P assembly with bulk and single molecule fluorescence kinetic studies. RPP-mediated alterations in RPR conformational sampling will be studied using fluorescence resonance energy transfer, and changes in RPR topology will be uncovered with small angle x-ray scattering and native mass spectrometry. Although activity versus fidelity tradeoffs have shaped the adaptive landscape of many enzymes, we expect our work to provide insights into how multiple RPPs allowed archaeal/eukaryotic RNase P to maintain robust cleavage without compromising processing fidelity on a broad range of substrates. This study will contribute to a framework for understanding the mechanistic basis of RNA-protein cooperation in RNPs and how dysfunctioning RNPs lead to disease.

概括我们的科学目标是了解蛋白质如何调节核糖核蛋白 (RNP) 的功能通过对其相关催化 RNA 进行结构改变来改变酶的活性，这一目标与公众高度相关。由于人们越来越认识到 RNP 在组织复杂性和人类疾病中的作用。提案中，我们将使用 RNase P 作为模型来测试我们的假设，即 RNP 的多功能性是由于蛋白质 - 尽管 RNase P 的主要功能是 5′′-成熟。前体 tRNA，最近的研究结果表明其功能使命已扩大，其中包括真核非编码 RNA 真核和古菌 RNase P 由催化 RPR (RNase P RNA) 和多个（4-10）RPP（RNase P 蛋白），与简单的细菌版本（1 RPR + 1 RPP）不同，因为所有。 RPR 在体外具有自身活性，但我们发现是否需要多种古菌和真核 RPP 尚不清楚。从古菌 RNase P 的逐步重建中，其组装中间体包含部分套件五个 RPP 和 RPR 表现出单独 RPR 或完整全息之间的处理活性和保真度。这些发现激发了我们的中心假设：RPP 与特定的结合。 RPR 区域独立和共同介导 RNA 结构变化，这些变化对于组装和组装至关重要。我们将通过两个具体目标来解决这个假设，以描述结构与功能的关系。组装完整 RNP 的中间体：(1) 剖析不同作用的结构基础古细菌 RPP 有助于 RPR 催化，(2) 绘制古细菌 RPP 在 RPR 上的组装图谱。为了研究 RPP 如何引导 RPR 达到其功能状态，我们提出了一种创新的位点组合：与直接功能读数相结合的特定和全局结构方法在目标 1 中，我们将探索古菌。使用 SHAPE-Seq 在核苷酸分辨率下由不同 RPP 套件诱导的 RPR 结构变化（通过引物延伸测序分析选择性2ʹ'-羟基酰化），一种高通量探测方法将引导来自 SHAPE-Seq 的 RNA 结构推论，将结构变化与功能结果联系起来。通过从束缚核酸酶作图获得并使用 RPR 测定进行验证的 RNA-蛋白质接触位点在目标 2 中，我们将调查 RNase P 组装过程中的层次结构和协作。 RPP 介导的 RPR 构象采样变化的分子荧光动力学研究。使用荧光共振能量转移进行研究，并且将揭示 RPR 拓扑的变化尽管活性与保真度之间存在权衡，但小角度 X 射线散射和天然质谱分析仍然存在。塑造了许多酶的适应性景观，我们希望我们的工作能够深入了解多重酶如何 RPP 使古菌/真核 RNase P 能够在不影响加工的情况下保持稳健的切割这项研究将有助于建立一个理解框架。 RNP 中 RNA-蛋白质合作的机制基础以及功能失调的 RNP 如何导致疾病。