Mechanistic investigations on the role of the ribosome-bound chaperones RAC and Ssb during nonstop- and polylysine protein expression

该项目的研究结果表明，核糖体结合的新生链可以通过先前未被发现的脱落机制释放，这导致在a位点具有意义密码子的核糖体上过早翻译终止（PMT）。我们发现，当核糖体在翻译聚赖氨酸编码序列时停顿，并且在核糖体结合的伴侣Ssb/RAC缺失的情况下，过早终止被强烈增强。相反，当翻译终止因子eRF3的浓度较低或缺少小核糖体蛋白Asc1时，早期翻译终止会大大减少，Asc1是核糖体相互作用蛋白（包括信号激酶）的枢纽。我们发现E3泛素连接酶Hel2以依赖Asc1的方式与核糖体相互作用，然后泛素化靠近Asc1的核糖体蛋白。作为该项目的延续，我们现在希望证实在意义密码子处过早翻译终止的机制，并了解Ssb/RAC阻止而Asc1/Hel2促进核糖体脱落的作用。为此，我们将以酵母为模型，运用生物化学和细胞生物学方法，包括体内易发生失速的报告蛋白分析、体内蛋白质-蛋白质接近试验、泛素化试验和酵母体外翻译系统，研究翻译失速的机制、关键因子需求和失速产物的命运。利用这些工具，我们将识别易导致翻译过早终止的密码子和序列上下文，并识别导致这种翻译错误的翻译终止因子突变体。我们将确定准确的翻译终止是否受到小亚基核糖体蛋白泛素化的调节，以及核糖体蛋白泛素化是否可逆和动态调节。我们将进一步通过实验测试Asc1/ hel2依赖性小核糖体亚基蛋白泛素化的可能性，该泛素化在Asc1在核糖体失速和药物诱导的核糖体毒性应激信号传导中已建立但看似不连贯的功能之间提供了联系。后者将涉及筛选激酶，在核糖体毒性应激时以Asc1/ hel2依赖的方式招募到核糖体。识别导致翻译终止错误的难翻译核苷酸序列、翻译因子缺陷和应激条件，将有助于我们进一步了解确保翻译准确性的基本机制。这项研究将增强我们对真核生物翻译及其与细胞应激反应和质量控制网络的整合的理解。

Findings of project revealed that ribosome-bound nascent chains can be released by a previously unappreciated drop-off mechanism, which leads to premature translation termination (PMT) on ribosomes with a sense codon in the A-site. We found that premature termination is strongly enhanced when ribosomes stall during translation of polylysine encoding sequences and in the absence of the ribosome-bound chaperones Ssb/RAC. In contrast, premature translation termination is strongly diminished when the concentration of the translation termination factor eRF3 is low, or in the absence of the small ribosomal protein Asc1, which serves as a hub for ribosome-interacting proteins, including signaling kinases. We found that the E3 ubiquitin ligase Hel2 interacts with the ribosome in an Asc1-dependent manner and then ubiquitinates ribosomal proteins in close proximity of Asc1.As a continuation of the project we now wish to corroborate the mechanism of premature translation termination at sense codons and understand the action by that Ssb/RAC prevents, while Asc1/Hel2 promotes ribosome drop-off. To that end, we will use yeast as a model and apply biochemical and cell biological methods including in vivo analysis of stalling-prone reporters, in vivo protein-protein proximity assays, ubiquitination assays, and a yeast in vitro translation system, to study the mechanism of translational stalling, key factor requirements, and the fate of stalling products. With these tools we shall identify the codons and sequence context, which is prone to premature translation termination and identify translation termination factor mutants, which promote this translational error. We will determine if accurate translation termination is regulated by the ubiquitination of small subunit ribosomal proteins and if ribosomal protein ubiquitination is reversible and dynamically regulated. We will further experimentally test the possibility that Asc1/Hel2-dependent ubiquitination of small ribosomal subunit proteins provides a link between the well established, but seemingly incoherent, functions of Asc1 in ribosome stalling and drug-induced ribotoxic stress signaling. The latter will involve a screen for kinases, recruited to ribosomes upon ribotoxic stress in an Asc1/Hel2-dependent manner.Recognition of difficult to translate nucleotide sequences, translation factor defects, and stress conditions, which induce translation termination errors will further our understanding of the essential mechanisms, which assure accuracy of translation. The study shall enhance our understanding of eukaryotic translation and its integration into the cellular stress response and quality control networks.