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

新合成蛋白质的折叠对所有细胞的功能至关重要。它由一个分子伴侣网络支持，其中一些分子伴侣通过参与翻译的核糖体和新生的链起到共翻译的作用。为了有效地支持折叠过程并协调伴侣活性与其他蛋白质成熟因子的作用，初生链与初生链的接触必须与翻译的进程精确同步。该项目旨在通过研究酿酒酵母中核糖体相关的伴侣三联体的功能，揭示蛋白质翻译和折叠机制协调的机制原理。这个三联体由Hsp70分子伴侣Ssb1，2(SSB)和由Hsp40分子伴侣Zuo1和Hsp70分子伴侣Ssz1组成的异构体Rac复合体组成。以选择性核糖体分析(SERP)为主要实验手段，我们将在体内以近密码子分辨率鉴定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，我们将确定介导RAC结合的新生链特征。在野生型和伴侣突变株中的SERP，以及纯化的伴侣与翻译核糖体的体外结合研究，将有助于探索Rac和SSB的功能相互作用，并揭示Rac的新生链结合如何在体内促进SSB的作用。该提案的第二个主要目的是调查RAC功能与翻译的协调。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，即翻译延长的变化指导伴侣功能。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下核糖体的mRNA特异性翻译速度，并探索mRNAs上发生碰撞的核糖体(二体)作为局部核糖体暂停的指标。将伴侣结合谱与探索翻译动力学的数据相关联，将揭示翻译速度的变化是否与核糖体的SSB和RAC作用相关。为了评估协调翻译速度和伴侣功能的机制，我们将测试RAC结合对tRNA结合和翻译延伸的可能直接影响，并调查核糖体停顿是否可能促进伴侣与翻译核糖体的联系。总之，这些分析将为协调Hsp70辅助的共翻译蛋白质折叠和翻译的复杂机制提供新的见解，并提高我们对蛋白质成熟与合成的概念理解。

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.