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

核糖体结合伴侣 RAC 和 Ssb 在不间断和多聚赖氨酸蛋白表达过程中作用的机制研究

基本信息

批准号：
244586127
负责人：
Professorin Dr. Sabine Karola Rospert
金额：
--
依托单位：
Institut für Biochemie und Molekularbiologie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2013
资助国家：
德国
起止时间：
2012-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/244586127?language=en
关键词：
Mechanistic investigations role ribosome bound

项目摘要

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.

该项目的研究结果表明，核糖体结合的新生链可以通过一种以前未被认识到的脱落机制释放，这导致核糖体上A位点有义密码子的过早翻译终止（PMT）。我们发现，提前终止强烈增强时，核糖体失速翻译过程中的多聚赖氨酸编码序列和核糖体结合的伴侣蛋白SSB/RAC的情况下。相反，当翻译终止因子eRF 3的浓度低时，或在不存在小核糖体蛋白Asc 1的情况下，过早的翻译终止会大大减少，Asc 1是核糖体相互作用蛋白（包括信号激酶）的中心。我们发现E3泛素连接酶Hel 2以Asc 1依赖的方式与核糖体相互作用，然后泛素化Asc 1附近的核糖体蛋白。作为该项目的继续，我们现在希望证实在有义密码子处提前终止翻译的机制，并了解Ssb/RAC阻止，而Asc 1/Hel 2促进核糖体脱落的作用。为此，我们将使用酵母作为模型，并应用生物化学和细胞生物学方法，包括在体内分析失速倾向的报告，在体内蛋白质-蛋白质邻近测定，泛素化测定和酵母体外翻译系统，研究翻译失速的机制，关键因素的要求，和失速产物的命运。有了这些工具，我们将确定的密码子和序列上下文，这是容易过早的翻译终止和识别翻译终止因子突变体，促进这种翻译错误。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的可能性，Asc 1/Hel 2依赖泛素化的小核糖体亚基蛋白提供了一个良好的，但似乎不连贯的，功能的Asc 1在核糖体失速和药物诱导的核糖毒性应激信号之间的联系。后者将涉及一个屏幕上的激酶，招募到核糖体上的核糖毒性压力在Asc 1/Hel 2依赖mannes.Recognition的困难翻译的核苷酸序列，翻译因子的缺陷，和应力条件，诱导翻译终止错误将进一步我们的理解的基本机制，保证翻译的准确性。这项研究将增强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中的理解。