Disentangling conformational and compositional heterogeneity

摘要 最近发现，“特化的”真核核糖体在某些核心区中富集或耗尽， 核糖体蛋白（RP）优先翻译mRNA序列组，而且这些组 mRNA的表达通常与特定的细胞途径有关（Shi等人，2017年）。这种核糖体与 组成的异质性和基因组调控已研究了质谱和测序 分析从不同核糖体群体分离的核糖体和mRNA序列。然而，简单 连接“特化”核糖体和序列偏好的规则尚未出现。我们的工作 合作者Maria Barna的实验室表明，RPS 25/eS 25（一种亚化学计量的RP）的存在或不存在， 位于真核生物核糖体40 S亚基mRNA出口通道附近，影响~150 基因.我们的假设是，这个核心RP通过改变mRNA的结构和结构来赋予mRNA识别。 核糖体的构象动力学。为了解决这个问题，我们将分离和表征核糖体， 有或没有RPS 25/eS 25通过cryoEM。 由于其基本上是单颗粒性质，cryoEM是检查大颗粒的最佳方法。 异质高分子机器然而，目前冷冻EM分析的最终结果是单一的， 结构反映了所有贡献构象的平均值。我们建议发展一类新的模型 它将动态结构的概念与构象异质性结合起来。我们将合作 与cisTEM的开发人员一起参数化结构模型的灵活性并迭代地细化模型 在cryoEM图的细化过程中沿着正则结构的运动。我们将完善灵活的模型， 使用这种新的功能性，有和没有RPS 25/eS 25的核糖体可以发现结构和功能之间的联系。 功能，揭示了翻译特异性的原子机制，并暗示了一个通用的机制， 核糖体组成的基因组调控。 总之，我们将为cryoEM开发编码结构灵活性的新结构细化功能 并将其应用于发现核糖体对不同mRNA的偏好之间的柔性差异 序列的这项研究将是一个原则的证明，为发现生物相关的结构运动， 单颗粒cryoEM，以及扩展的核糖体和mRNA模型将表明基因组的机制， 在蛋白质合成阶段的调节。

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.