Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa-shi, Chiba 277-8562, Japan.
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
J Biochem. 2020 May 1;167(5):451-462. doi: 10.1093/jb/mvaa022.
We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.
我们最近开发了一种体外酵母重组翻译系统，该系统能够合成长多肽。利用该系统，我们研究了 eIF5A 及其 hypusine 修饰在翻译长开放阅读框内的多聚脯氨酸序列中的作用。我们发现，插入蛋白质内部位置的多聚脯氨酸基序仅在低 Mg2+浓度下阻止翻译，肽酰基多聚脯氨酸-tRNA 内在地使 80S 核糖体不稳定。我们证明未修饰的 eIF5A 基本上可以解决这种核糖体停滞问题；然而，hypusine 修饰极大地刺激了 eIF5A 拯救多聚脯氨酸介导的核糖体停滞的能力，对于有效翻译 N 末端或长内部多聚脯氨酸基序尤为重要。
