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

研究发现，核糖体结合的初生链可以通过一种以前未被发现的脱落机制被释放，这会导致A位有义密码子的核糖体提前翻译终止(PMT)。我们发现，当核糖体在多赖氨酸编码序列的翻译过程中停滞时，以及在缺乏核糖体结合的伴侣蛋白SSB/RAC的情况下，提前终止被强烈地增强。相反，当翻译终止因子eRF3的浓度较低时，或者在缺乏小的核糖体蛋白Asc1的情况下，过早的翻译终止会大大减少，Asc1是核糖体相互作用蛋白的枢纽，包括信号转导酶。我们发现E3泛素连接酶Hel2以Asc1依赖的方式与核糖体相互作用，然后泛化Asc1附近的核糖体蛋白。作为项目的继续，我们现在希望证实在正义密码子提前终止翻译的机制，并了解SSB/RAC防止的作用，而Asc1/Hel2促进核糖体下降。为此，我们将以酵母为模型，应用生化和细胞生物学方法，包括体内易失速记者的分析、体内蛋白质-蛋白质接近分析、泛素化分析和酵母体外翻译系统，以研究翻译失速的机制、关键因素要求和失速产品的命运。使用这些工具，我们将识别密码子和序列上下文，这容易过早地终止翻译，并识别导致这种翻译错误的翻译终止因子突变。我们将确定准确的翻译终止是否受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。我们将进一步实验测试Asc1/Hel2依赖的核糖体小亚基蛋白泛素化在Asc1在核糖体停滞中的功能和药物诱导的核毒应激信号之间提供联系的可能性。Asc1在核糖体停滞中的功能已经确立，但似乎不连贯。后者将涉及在核毒应激下以Asc1/Hel2依赖的方式招募到核糖体的激酶的筛选。识别难以翻译的核苷酸序列、翻译因子缺陷和导致翻译终止错误的应激条件将进一步加深我们对确保翻译准确性的基本机制的理解。这项研究将加深我们对真核翻译及其整合到细胞应激反应和质量控制网络的理解。

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.