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。我们最近已经证明，当翻译蛋白含有连续的多聚脯氨酸延伸时，细菌核糖体会被阻止，这种停止可以被翻译延伸因子EF-P缓解。此外，我们还发现Ef-P中的溶酶34在翻译后被YjeA和YjeK酶降解，而Shewanella EF-P中的精氨酸32被EARP催化鼠李糖化，这对Ef-P的解救活性是至关重要的。许多毒力因子含有多聚脯氨酸延伸段，这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，但在许多细菌门中都不存在YjeA和YjeK或Earp修饰酶，尽管这些细菌编码大量的含有多脯氨酸的蛋白质，因此表明存在新的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，特别是包含临床上重要的致病菌的门，如Firmicius(例如葡萄球菌和肠球菌)和放线杆菌(例如分枝杆菌)。此外，我们建议应用体内筛选试验来鉴定低分子EF-P抑制剂，以开发新的抗菌剂。最后，我们的目标是解析修饰的EF-P结合到多聚脯氨酸停滞的核糖体上的结构，以阐明多脯氨酸伸展停滞核糖体的机制以及EF-P如何缓解翻译停滞。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性和机械性的见解，而且还将扩大翻译后修改系统的总体范围。

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.