Department of Molecular Genetics, Ohio State University, Columbus, Ohio, USA.
Center for RNA Biology, Ohio State University, Columbus, Ohio, USA.
mBio. 2018 Apr 3;9(2):e00306-18. doi: 10.1128/mBio.00306-18.
Elongation factor P (EF-P) is a ubiquitous translation factor that facilitates translation of polyproline motifs. In order to perform this function, EF-P generally requires posttranslational modification (PTM) on a conserved residue. Although the position of the modification is highly conserved, the structure can vary widely between organisms. In , EF-P is modified at Lys32 with a 5-aminopentanol moiety. Here, we use a forward genetic screen to identify genes involved in 5-aminopentanolylation. Tandem mass spectrometry analysis of the PTM mutant strains indicated that , , and are required for modification and that , , and influence the level of modification. Structural analyses also showed that EF-P can retain unique intermediate modifications, suggesting that 5-aminopentanol is likely directly assembled on EF-P through a novel modification pathway. Phenotypic characterization of these PTM mutants showed that each mutant does not strictly phenocopy the mutant, as has previously been observed in other organisms. Rather, each mutant displays phenotypic characteristics consistent with those of either the mutant or wild-type depending on the growth condition. polyproline reporter data indicate that the observed phenotypic differences result from variation in both the severity of polyproline translation defects and altered EF-P context dependence in each mutant. Together, these findings establish a new EF-P PTM pathway and also highlight a unique relationship between EF-P modification and polyproline context dependence. Despite the high level of conservation of EF-P, the posttranslational modification pathway that activates EF-P is highly divergent between species. Here, we have identified and characterized in a novel posttranslational modification pathway. This pathway not only broadens the scope of potential EF-P modification strategies, but it also indicates that EF-P modifications can be assembled directly on EF-P. Furthermore, characterization of these PTM mutants has established that an altered modification state can impact both the severity of polyproline translational defects and context dependence.
延伸因子 P（EF-P）是一种普遍存在的翻译因子，可促进多脯氨酸基序的翻译。为了执行此功能，EF-P 通常需要对保守残基进行翻译后修饰（PTM）。尽管修饰的位置高度保守，但结构在不同生物体之间差异很大。在 中，EF-P 在赖氨酸 32 位上被 5-氨基戊醇修饰。在这里，我们使用正向遗传筛选来鉴定参与 5-氨基戊醇化的基因。对 PTM 突变株的串联质谱分析表明， 、 和 是修饰所必需的， 和 影响修饰水平。结构分析还表明，EF-P 可以保留独特的中间修饰，这表明 5-氨基戊醇可能通过一种新的修饰途径直接组装在 EF-P 上。这些 PTM 突变体的表型特征表明，每个突变体并不严格模拟 突变体，正如以前在其他生物体中观察到的那样。相反，每个突变体根据生长条件显示出与 突变体或野生型 一致的表型特征。多脯氨酸报告基因数据表明，观察到的表型差异是由于每个突变体中多脯氨酸翻译缺陷的严重程度以及 EF-P 上下文依赖性的变化。这些发现共同建立了一个新的 EF-P PTM 途径，并强调了 EF-P 修饰与多脯氨酸上下文依赖性之间的独特关系。尽管 EF-P 的高度保守，但激活 EF-P 的翻译后修饰途径在物种之间高度不同。在这里，我们在 中鉴定并表征了一种新的翻译后修饰途径。该途径不仅拓宽了潜在 EF-P 修饰策略的范围，而且表明 EF-P 修饰可以直接组装在 EF-P 上。此外，这些 PTM 突变体的特性表明，修饰状态的改变会影响多脯氨酸翻译缺陷的严重程度和上下文依赖性。
