Dissecting functional cooperation among subunits in a catalytic ribonucleoprotein

总结 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 酶通过结构变化对其相关的催化RNA。这一目标与公众高度相关。 由于RNP在组织复杂性和人类疾病中的作用日益受到重视，在这 建议，我们将使用RNase P作为模型来测试我们的假设，即RNPs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 ′端成熟， 前体tRNA，最近的研究结果表明，一个扩展的功能使命，包括生物起源的 真核生物非编码RNA。真核和古细菌RNase P由催化RPR（RNase P RNA）和 多个（4-10）RPP（RNase P蛋白），不同于简单的细菌版本（1 RPR + 1 RPP）。因为所有 RPR本身在体外是有活性的，对多种古细菌和真核生物RPP的需求尚不清楚。我们发现 从古细菌RNase P的逐步重建，其组装中间体包括部分套件， 五个RPPs和RPR在单独的RPR或完整的全息图之间表现出处理的活性和保真度。 酶（RPR +所有RPP）。这些发现激发了我们的中心假设，即RPPs与特异性 RPR区域独立地和共同地介导RNA结构变化， 催化作用我们将通过两个具体的目的来阐述这一假设，以描述结构-功能关系， 中间体组装完整的RNP：（1）解剖的不同作用的结构基础， 帮助RPR催化的古细菌RPPs，和（2）绘制古细菌RPPs在RPR上的组装景观。 为了研究RPR如何引导RPR进入其功能状态，我们提出了一种创新的网站组合， 具体的和全球的结构方法耦合到直接功能读出。在目标1中，我们将探索古细菌 在使用SHAPE-Seq的核苷酸解析中由不同RPPs套件诱导的RPR结构变化 （通过引物延伸测序分析的选择性2 ′-羟基酰化），这是一种高通量的方法来探测 RNA结构从SHAPE-Seq推断，将结构变化与功能结果联系起来， 通过从拴系核酸酶图谱获得的RNA-蛋白质接触位点，并使用RPR测定进行验证， 变种人在目标2中，我们将调查RNase P组装过程中的层次和合作， 分子荧光动力学研究。RPR构象采样中的RPP介导的改变将被 研究使用荧光共振能量转移，并在RPR拓扑结构的变化将被发现与 小角X射线散射和自然质谱法。虽然活动与忠诚度的权衡 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关于多种酶如何适应环境的见解。 RPP允许古细菌/真核生物RNase P在不损害加工的情况下保持稳健的切割 在广泛的基底上的保真度。这项研究将有助于理解的框架， 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。

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.