Dissecting functional cooperation among subunits in a catalytic ribonucleoprotein

摘要 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。 酶通过与其相关的催化核糖核酸的结构变化。这一目标与公众高度相关 由于人们越来越多地认识到RNPs在组织复杂性和人类疾病中的作用，我们已经开始关注RNPs对人类健康的影响。在这 提议，我们将使用RNaseP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- 介导了它们的RNA核心的结构变化。尽管核糖核酸酶P的主要功能是5ʹ‘-成熟 前体tRNAs，最近的发现表明一个扩大的功能使命，包括生物发生 真核非编码RNA。真核生物和古生物的RNaseP由催化的RPR(RNaseP RNA)和 多个(4-10)RPP(RNaseP蛋白)，不同于更简单的细菌版本(1RPR+1RPP)。因为所有人 RPR在体外具有自身的活性，但是否需要多种古生菌和真核RPP尚不清楚。我们发现 来自古生菌RNaseP的逐步重组，它的组装中间产物包括部分 五个RPP和RPR在单独的RPR或完全全息之间显示出处理的活性和保真度- 酶(RPR+ALL RPP)。这些发现激发了我们的中心假设，即RPP与特定的 RPR区域独立地和共同地介导对组装和 催化作用。我们将用两个具体的目标来解决这一假说，以描绘出结构与功能的关系 中间体在组装完整的RNP的过程中：(1)剖析了不同角色的结构基础 古生菌RPP在辅助RPR催化中的作用，以及(2)在RPR上绘制古生菌RPP的组装景观。 为了研究RPP如何引导RPR走向其功能状态，我们提出了一个创新的站点组合- 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，我们将探索古生物 用SHAPE-SEQ方法研究核苷酸拆分时不同RPP组引起的RPR结构变化 (引物延伸测序分析选择性2ʹ‘-羟基酰化)，一种高通量的探针方法 RNA结构。SHAPE-SEQ的推论，将结构变化与功能结果联系起来，将得到指导 通过系留核酸酶图谱获得的RNA-蛋白质接触位点，并通过RPR分析进行验证 变种人。在目标2中，我们将考察RNaseP组装过程中的层次结构和协作关系 分子荧光动力学研究。RPP介导的RPR构象采样的变化将是 利用荧光共振能量转移进行研究，RPR拓扑结构的变化将被揭示 小角x射线散射和自然质谱学。尽管活动与保真度之间的权衡 塑造了许多酶的适应格局，我们希望我们的工作能提供对多个 RPP允许古生物/真核生物RNaseP在不影响加工的情况下保持强劲的切割 在广泛的基板上实现保真度。这项研究将有助于建立一个框架来理解 RNPs中RNA-蛋白质合作的机制基础以及功能失调的RNPs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.