Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.
Reverse genetics-based biochemical studies of the ribosomal exit tunnel constriction region in eukaryotic ribosome stalling: spatial allocation of the regulatory nascent peptide at the constriction.
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基于反向遗传学的真核核糖体停滞中核糖体出口隧道收缩区域的生化研究：收缩处调节新生肽的空间分配。
DOI：
10.1093/nar/gkz1190
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Satoshi Naito
中科院分区：
文献类型：
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作者：
Seidai Takamatsu;Yubun Ohashi;Noriyuki Onoue;Yoko Tajima;Tomoya Imamichi;Shinya Yonezawa;Kyoko Morimoto;Hitoshi Onouchi;Yui Yamashita;Satoshi Naito
许多调节性新生肽已被证明通过在翻译期间引起程序化核糖体停滞来调节基因表达。新生肽通过出口通道从核糖体中出现，并且沿着核糖体蛋白uL 4和uL 22的β环结构突出到通道中以形成收缩区的路径的沿着的三分之一。结构研究表明新生肽与包括收缩区在内的出口通道组分之间存在相互作用。然而，在真核生物中，缺乏对收缩区参与核糖体停滞的遗传学研究。在这里，我们建立了在uL4的β环结构中携带突变的转基因拟南芥系。使用来自携带突变核糖体的转基因拟南芥的无细胞翻译系统的翻译分析表明，uL4突变减少了四种真核生物失速系统的核糖体失速，包括那些失速结构已经解决的系统。我们的数据，这表明不同的uL4突变的影响取决于失速系统，解释了空间分配的新生肽的收缩，推导出的结构研究。相反，我们的数据可以预测分配的新生肽在收缩的失速系统的结构研究没有完成。
A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which β-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the β-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.