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）的功能酶通过其相关催化RNA的结构变化。这个目标与公众高度相关由于人们对RNP在组织复杂性和人类疾病中的作用的增长越来越多。在这个提案，我们将使用RNase P作为模型来测试我们的假设RNP的多功能性是由于蛋白质 - 介导的RNA核心结构变化。虽然RNase P的主要功能是5'的成熟前体TRNA，最近的发现表明了一个扩展的功能任务，其中包括生物发生真核非编码RNA。真核和古细菌RNase P由催化RPR（RNase P RNA）和与简单的细菌版本（1 RPR + 1 RPP）不同，多个（4-10）RPP（RNase P蛋白）。因为全部 RPR在体外很活跃，需要多个古细菌和真核RPP的需求尚不清楚。我们发现从统计的古细菌RNASE P的逐步重构，其组装中间可以完成部分套件五个RPP和RPR暴露的活动和仅在RPR之间或完整的全体性之间的加工忠诚度酶（RPR +所有RPP）。这些发现激发了我们的中心假设，即RPP结合了特定 RPR区域独立介导了RNA结构的变化，对组装至关重要催化。我们将以两个具体的目的来解决这一假设中间体在组装完整RNP的途中：（1）剖析结构基础的不同作用在RPR催化中的古细菌RPP，（2）在RPR上绘制古细菌RPP的组装景观。为了研究RPP如何指导RPR达到其功能状态，我们提出了站点的创新组合特定的全球结构方法结合了直接功能读数。在AIM 1中，我们将探测古细菌 RPR结构变化在核苷酸分辨率下由不同的RPP套件诱导（通过引物扩展测序分析的选择性2ʹ'-羟基酰基分析），一种高吞吐量的方法 RNA结构。 Shape-seq的推论，将结构变化与功能结果联系起来，将被指导通过从螺旋核酸酶映射获得的RNA-蛋白接触位点，并使用RPR测定进行验证突变体。在AIM 2中，我们将调查RNase P组装期间的层次结构与合作分子荧光动力学研究。 RPP介导的RPR构象采样的改变将是使用荧光共振能量转移进行研究，RPR拓扑的变化将被发现小角度X射线散射和天然质谱法。尽管活动与忠诚的权衡有塑造了许多酶的适应性景观，我们希望我们的工作能够提供有关多重多重方式的见解 RPP允许古细胞/真核RNase P保持稳健的裂解而不会损害处理在广泛的底物上的保真度。这项研究将有助于理解 RNP中RNA-蛋白合作的机理基础以及功能障碍RNP如何导致疾病。