Schuller Anthony P, Wu Colin Chih-Chien, Dever Thomas E, Buskirk Allen R, Green Rachel
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Mol Cell. 2017 Apr 20;66(2):194-205.e5. doi: 10.1016/j.molcel.2017.03.003. Epub 2017 Apr 6.
The eukaryotic translation factor eIF5A, originally identified as an initiation factor, was later shown to promote translation elongation of iterated proline sequences. Using a combination of ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in elongation and uncover a critical function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show ribosome accumulation at stop codons and in the 3' UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted translation system, we find that eIF5A strongly promotes the translation of the stalling sequences identified by profiling and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in elongation and termination, rationalizing its high cellular abundance and essential nature.
真核生物翻译因子eIF5A最初被鉴定为起始因子，后来发现它能促进重复脯氨酸序列的翻译延伸。通过核糖体分析和体外生物化学相结合的方法，我们报道了eIF5A在延伸过程中具有更广泛的作用，并揭示了eIF5A在终止过程中的关键功能。对eIF5A缺失菌株的核糖体分析揭示了一个全局性的延伸缺陷，大量核糖体在许多序列处停滞，不限于脯氨酸序列。我们的数据还显示核糖体在终止密码子处和3'UTR区域积累，这表明在缺乏eIF5A的情况下终止过程存在全局性缺陷。使用体外重构的翻译系统，我们发现eIF5A强烈促进通过分析鉴定出的停滞序列翻译，并使肽基-tRNA水解速率提高超过17倍。我们得出结论，eIF5A在延伸和终止过程中广泛发挥作用，这解释了其在细胞中高丰度和必需性的原因。
