Disentangling conformational and compositional heterogeneity

摘要 最近发现，真核细胞中“特化”的核糖体在某些核心富含或枯竭。 核糖体蛋白(RP)优先翻译成组的mRNA序列，而且这些组 MRNAs的一部分往往与特定的细胞通路​(Ship等人，2017年)​有关。核糖体之间的这种联系 通过质谱学和测序研究了组成的异质性和基因组的调节。 分离自不同核糖体群体的核糖体和信使核糖核酸序列分析。然而，简单 将“专门的”核糖体和序列偏好联系起来的规则还没有出现。工作由我们的 合作者玛丽亚·巴纳的实验室表明，RPS25/ES25，一种亚化学计量比的RP的存在或不存在 位于真核生物核糖体40S亚基上的mRNA出口隧道附近，影响~150的翻译 基因。我们的假设是，这种核心RP通过改变结构和 核糖体的构象动力学。为了解决这个问题，我们将分离并鉴定核糖体。 通过CryoEM检测RPS25/ES25的存在与否。 由于其基本的单粒子性质，低温电磁是检查大型 多相大分子机器。然而，目前低温电子显微镜分析的最终结果是单一的 反映所有贡献构象的平均值的结构。我们建议开发一类新的模型 这包含了具有构象异质性的动态结构的概念。我们将合作 与cisTEM的开发人员一起对结构模型的柔度进行参数化，并迭代细化模型 在冷冻EM图精化过程中沿正则结构的运动。我们将细化灵活的模式 带有和不带有RPS25/ES25的核糖体使用这一新功能来发现结构和 功能，揭示了翻译专一性的原子机制，并暗示了 核糖体组成对基因组的调控。 总之，我们将为CryoEM开发新的结构优化功能，以编码结构灵活性 并将其应用于发现优先于不同mrna的核糖体之间柔韧性的差异。 序列。这项研究将是发现与生物相关的结构运动的原则证明 单颗粒低温电子显微镜，以及扩展的核糖体和mRNA模型将为基因组提供一种机制 蛋白质合成阶段的调控。

ABSTRACT It has recently been discovered that “specialized” eukaryotic ribosomes enriched or depleted in certain core ribosomal proteins (RPs) preferentially translate groups of mRNA sequences, and moreover that these groups of mRNAs often relate to specific cellular pathways ​(Shi et al., 2017)​. This link between ribosome compositional heterogeneity and genome regulation has been studied by mass spectrometric and sequencing analyses of ribosomes and mRNA sequences isolated from distinct ribosome populations. However, simple rules linking “specialized” ribosomes and sequence preferences have not yet emerged. Work by our collaborator Maria Barna’s lab showed that the presence or absence of RPS25/eS25, a substoichiometric RP located near the mRNA exit tunnel on the 40S subunit of the eukaryotic ribosome, affects translation of ~150 genes. Our hypothesis is that this core RP imparts recognition of mRNAs by altering the structure and conformational dynamics of the ribosome. To address this question, we will isolate and characterize ribosomes with or without RPS25/eS25 by cryoEM. Due to its fundamentally single particle nature, cryoEM is an optimal method for examining large heterogeneous macromolecular machines. However, at present the end result of cryoEM analysis is a single structure reflecting the average of all contributing conformations. We propose to develop a new class of model that incorporates the concept of a dynamic structure with conformational heterogeneity. We will collaborate with the developers of cisTEM to parameterize flexibility of a structural model and to iteratively refine model motions alongside the canonical structure during refinement of cryoEM maps. We will refine flexible models of ribosomes with and without RPS25/eS25 using this new functionality to discover a link between structure and function, revealing the atomic mechanisms of translation specificity and implicating a general mechanism for genome regulation by ribosome composition. In summary, we will develop new structure refinement capabilities for cryoEM that encode structural flexibility and apply them to the discovery of differences in flexibility between ribosomes preferential to different mRNA sequences. This study will be a proof of principle for the discovery of biologically relevant structural motions by single particle cryoEM, and the expanded ribosome and mRNA models will suggest a mechanism for genome regulation at the stage of protein synthesis.