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

点击翻译按钮获取中文摘要

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.