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首页> 外文期刊>The FEBS journal >Synthetic post-translational modifications of elongation factor P using the ligase EpmA
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Synthetic post-translational modifications of elongation factor P using the ligase EpmA

机译:利用连接酶EPMA的伸长率伸长率伸缩性改性的合成翻译改性

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摘要

Canonically, tRNA synthetases charge tRNA. However, the lysyl-tRNA synthetase paralog EpmA catalyzes the attachment of (R)-beta-lysine to the epsilon-amino group of lysine 34 of the translation elongation factor P (EF-P) inEscherichia coli. This modification is essential for EF-P-mediated translational rescue of ribosomes stalled at consecutive prolines. In this study, we determined the kinetics of EpmA and its variant EpmA_A298G to catalyze the post-translational modification of K34 in EF-P with eight noncanonical substrates. In addition, acetylated EF-P was generated using an amber suppression system. The impact of these synthetically modified EF-P variants onin vitrotranslation of a polyproline-containing NanoLuc luciferase reporter was analyzed. Our results show that natural (R)-beta-lysylation was more effective in rescuing stalled ribosomes than any other synthetic modification tested. Thus, our work not only provides new biochemical insights into the function of EF-P, but also opens a new route to post-translationally modify proteins using EpmA.
机译:典型地,tRNA合成酶对tRNA负责。然而,赖氨酰-tRNA合成酶paralog EpmA催化(R)-β-赖氨酸附着到大肠杆菌翻译延伸因子P(EF-P)赖氨酸34的ε-氨基上。这种修饰对于EF-P介导的连续脯氨酸滞留核糖体的翻译拯救至关重要。在这项研究中,我们测定了EpmA及其变体EpmA_A298G在八种非标准底物下催化EF-P中K34的翻译后修饰的动力学。此外,使用琥珀色抑制系统生成乙酰化EF-P。分析了这些人工修饰的EF-P变体对含聚脯氨酸的纳米Luc荧光素酶报告基因的体外翻译的影响。我们的结果表明,天然(R)-β-赖氨酸化在挽救停滞的核糖体方面比任何其他经测试的合成修饰更有效。因此,我们的工作不仅为EF-P的功能提供了新的生化见解,而且为使用EpmA对蛋白质进行翻译后修饰开辟了新途径。

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