Coordination of mRNA translation with the action of ribosome-associated chaperones in yeast

新合成的蛋白质的折叠对于所有细胞的功能都是至关重要的。它由分子伴侣网络支持，其中一些通过接合翻译核糖体和新生链协同作用。为了有效地支持折叠过程并协调分子伴侣活性与其他蛋白质成熟因子的作用，新生链与新生链的接合必须与翻译的进展精确同步。本项目旨在通过研究S.啤酒。该三联体由Hsp70分子伴侣Ssb 1，2（Ssb）和Hsp40分子伴侣Zuo 1与Hsp70分子伴侣Ssz 1组成的异聚体RAC复合物组成。我们将利用选择性核糖体分析技术（Selective Ribosome Profiling，SeRP），在体内近密码子分辨下鉴定RAC的新生链相互作用体。通过对RAC相互作用谱进行生物信息学分析，我们将鉴定介导RAC结合的新生链特征。SeRP在野生型和伴侣蛋白突变株，支持在体外结合研究纯化的伴侣蛋白与翻译核糖体，将有助于探索RAC和SSB的功能相互作用，并揭示如何新生链结合RAC有助于在体内的行动SSB。该提案的第二个主要目标是研究RAC职能与翻译的协调。我们将测试RAC结合是否以及如何影响翻译动力学，并测试替代模型，即翻译延伸的变化引导分子伴侣功能。我们将进行径流核糖体分析，以确定野生型和伴侣突变株背景中核糖体的mRNA特异性翻译速度，并探索mRNA上碰撞核糖体（二体）的发生率作为局部核糖体暂停的指标。将伴侣蛋白结合谱与探索翻译动力学的数据相关联将揭示翻译速度变化是否与核糖体上的Ssb和RAC作用相关。为了评估协调翻译速度和伴侣蛋白功能的机制，我们将测试RAC结合对tRNA结合和翻译延伸的可能直接影响，并研究由mRNA或新生链内的特征赋予的核糖体暂停是否可能促进伴侣蛋白与翻译核糖体的缔合。这些分析将提供新的见解，复杂的机制，协调热休克蛋白70协助共同，翻译蛋白质折叠与翻译，提高我们的概念性理解蛋白质成熟是如何耦合到合成。

Folding of newly synthesized proteins is of fundamental importance for the functioning of all cells. It is supported by a network of molecular chaperones, some of which act co-translationally by engaging the translating ribosome and the nascent chain. To efficiently support the folding process and coordinate chaperone activity with the action of other protein maturation factors, nascent chain engagement with nascent chains must be precisely synchronized with the progression of translation. The proposed project aims to unravel mechanistic principles of the coordination of the protein translation and folding machineries, by studying the function of the ribosome associated chaperone triad in S. cerevisiae. This triad consists of the Hsp70 chaperone Ssb1,2 (Ssb) and the heteromeric RAC complex composed of the Hsp40 chaperone Zuo1 and the Hsp70 chaperone Ssz1.The first major aim of the proposal is to analyze the coordinative role of RAC on Ssb-mediated chaperoning of nascent polypeptides. Using Selective Ribosome Profiling (SeRP) as major experimental approach, we will identify the nascent chain interactome of RAC at near-codon resolution in vivo. By performing bioinformatic analysis of RAC interaction profiles we will identify nascent chain features that mediate RAC binding. SeRP in wild type and chaperone mutant strains, supported by in vitro binding studies of purified chaperones with translating ribosomes, will help exploring the functional interplay of RAC and Ssb and reveal how nascent chain binding of RAC contributes to the action of Ssb in vivo. The second major aim of the proposal investigates the coordination of RAC function with translation. We will test whether and how RAC binding impacts the kinetics of translation and also test the alternative model, that variation of translation elongation guides chaperone function. We will perform run-off ribosome profiling to determine mRNA-specific translation speed of ribosomes in wild type and chaperone mutant strain backgrounds and explore the prevalence of colliding ribosomes (disomes) on mRNAs as an indicator of local ribosome pausing. Correlating chaperone binding profiles with the data exploring translation kinetics will reveal whether translation speed changes correlate with Ssb and RAC action at the ribosome. To assess the mechanism coordinating translation speed and chaperone function, we will test a possible direct impact of RAC binding on tRNA binding and translation elongation and investigate whether ribosome pausing, conferred by features within mRNAs or nascent chains, may facilitate the association of chaperones with translating ribosomes.Together, these analyses will provide new insights into the intricate mechanism coordinating Hsp70- assisted co-translational protein folding with translation and improve our conceptual understanding how protein maturation is coupled to synthesis.