Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA
Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA
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DOI：
10.1038/sj.emboj.7601105
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发表时间：
2006-05-17
期刊：
影响因子：
11.4
通讯作者：
Suzuki, Tsutomu
中科院分区：
文献类型：
--
作者：
Noma, Akiko;Kirino, Yohei;Suzuki, Tsutomu
Wybutosine（yW）是一种三环核苷，在真核生物苯丙氨酸tRNA的反密码子附近的3 '-位发现具有大的侧链。yW通过在核糖体上解码期间稳定密码子-反密码子相互作用来支持密码子识别。为了确定基因负责yW合成的酿酒酵母的非特征基因，我们采用了系统的反向遗传方法结合质谱（“核糖核组分析”）。YPL 207 w、YML 005 w、YGL 050 w和YOL 141 w（分别命名为TYW 1、TYW 2、TYW 3和TYW 4）是yW合成所必需的基因。对yW的每个修饰中间体的质谱分析揭示了其顺序的生物合成途径。TYW 1是一种铁硫（Fe-S）簇蛋白，负责形成三环。从yW-187的多步酶促形成yW可以在体外使用重组TYW 2，TYW 3和TYW 4与S-腺苷甲硫氨酸重建，这表明yW合成可能通过由多个组分与前体tRNA组装形成的复合物中的顺序反应进行。植物直系同源物是由TYW 2和TYW 3与TYW 4的C-末端结构域组成的大融合蛋白的事实也支持该假设。
Wybutosine (yW) is a tricyclic nucleoside with a large side chain found at the 3 '-position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports codon recognition by stabilizing codon-anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry ('ribonucleome analysis'). Four genes YPL207w, YML005w, YGL050w and YOL141w (named TYW1, TYW2, TYW3 and TYW4, respectively) were essential for yW synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. TYW1 is an iron-sulfur (Fe-S) cluster protein responsible for the tricyclic formation. Multistep enzymatic formation of yW from yW-187 could be reconstituted in vitro using recombinant TYW2, TYW3 and TYW4 with S-adenosylmethionine, suggesting that yW synthesis might proceed through sequential reactions in a complex formed by multiple components assembled with the precursor tRNA. This hypothesis is also supported by the fact that plant ortholog is a large fusion protein consisting of TYW2 and TYW3 with the C-terminal domain of TYW4.