In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide
In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide
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DOI：
10.1093/jb/mvaa022
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发表时间：
2020-05-01
影响因子：
2.7
通讯作者：
Takeuchi-Tomita, Nono
中科院分区：
文献类型：
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作者：
Abe, Taisho;Nagai, Riku;Takeuchi-Tomita, Nono
我们最近开发了一个体外酵母重组翻译系统，它能够合成长的多肽。利用该系统，我们研究了eIF 5A及其羟腐胺赖氨酸修饰在长开放阅读框内翻译聚脯氨酸序列中的作用。我们发现，聚脯氨酸基序插入在蛋白质的内部位置逮捕翻译专门在低Mg 2+浓度，和peptidylpolyproline-tRNA本质上不稳定的80 S核糖体。我们证明，未修饰的eIF 5A基本上解决了这样的核糖体失速;然而，羟腐胺赖氨酸修饰大大刺激了eIF 5A拯救聚脯氨酸介导的核糖体失速的能力，并且对于N-末端或长的内部聚脯氨酸基序的有效翻译特别重要。
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.