Modification, inhibition and function of elongation factor EF-P

蛋白质合成的基本过程是在核糖体上催化的。我们最近证明，当翻译蛋白质含有连续的脯氨酸延伸时，细菌核糖体会被阻止，并且这种阻止会被翻译延伸因子EF-P缓解。此外，我们可以证明，大肠埃希氏菌EF-P翻译后被YjeA和YjeK酶对赖氨酸34的ß-赖氨酸化，以及希瓦氏菌EF-P被EarP酶对精氨酸32的鼠李糖基化，对EF-P的救援活性是至关重要的。许多毒力因子含有聚脯氨酸延伸，解释了为什么修饰的EF-P对细菌致病性至关重要。虽然EF-P是保守的，但在许多细菌门中不存在修饰酶YjeA和YjeK或EarP，尽管这些细菌编码大量含聚脯氨酸的蛋白质，这表明存在新的EF-P修饰途径。在这里，我们建议识别和表征这些新的修饰途径，特别关注含有临床重要致病菌的门，如厚壁菌门（如葡萄球菌和肠球菌）和放线菌门（如分枝杆菌）。此外，我们建议应用体内筛选试验来鉴定低分子量EF-P抑制剂，用于开发新的抗菌药物。最后，我们的目标是解析修饰的EF-P与聚脯氨酸停滞核糖体结合的结构，以阐明聚脯氨酸拉伸停滞核糖体的机制以及EF-P如何减轻翻译阻滞。总的来说，我们相信我们的建议不仅将为翻译的基本过程提供新的功能和机制见解，而且还将扩展翻译后修饰系统的范围。

The fundamental process of protein synthesis is catalyzed on ribosomes. We have recently demonstrated that bacterial ribosomes become arrested when translating proteins contain consecutive polyproline stretches, and that this arrest is alleviated by the translation elongation factor EF-P. Furthermore, we could show that the post-translational ß-lysinylation of lysine34 of Escherichia coli EF-P by the enzymes YjeA and YjeK and the rhamnosylation of arginine32 of Shewanella EF-P by EarP, respectively, are critical for the rescue activity of EF-P. Many virulence factors contain polyproline stretches, explaining why modified EF-P is critical for bacterial pathogenicity. While EF-P is conserved, the modification enzymes YjeA and YjeK or EarP are absent in many bacterial phyla, even though these bacteria encode a high number of polyproline-containing proteins, thus suggesting the existence of novel EF-P modification pathways. Here we propose to identify and characterize these novel modification pathways, with a particular focus on phyla containing clinically important pathogenic bacteria, such as Firmicutes (e.g., Staphylococcus and Enterococcus) and Actinobacteria (e.g., Mycobacterium). In addition, we propose to apply an in vivo screening assay to identify low molecular weight EF-P inhibitors for the development of novel antimicrobial agents. Lastly, we aim to resolve structures of modified EF-P bound to polyproline-stalled ribosomes in order to elucidate the mechanism by which polyproline stretches stall ribosomes as well as how EF-P alleviates the translational arrest. Collectively, we believe our proposal will not only provide new functional and mechanistic insights into the fundamental process of translation, but also extend the spectrum of post-translational modification systems in general.