In vitro trans-translation of Thermus thermophilus:: Ribosomal protein S1 is not required for the early stage of trans-translation
In vitro trans-translation of Thermus thermophilus:: Ribosomal protein S1 is not required for the early stage of trans-translation
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DOI：
10.1261/rna.363207
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发表时间：
2007-04-01
期刊：
影响因子：
4.5
通讯作者：
Himeno, Hyouta
中科院分区：
文献类型：
--
作者：
Takada, Kazuma;Takemoto, Chie;Himeno, Hyouta
传递信使RNA （Transfer-messenger RNA, tmRNA）在反式翻译中扮演tRNA和mRNA的双重角色，在反式翻译过程中，核糖体用编码标签肽的tmRNA取代mRNA。这些过程被认为涉及几种tmrna结合蛋白，包括SmpB和核糖体蛋白S1。为了研究反翻译的分子机制，我们利用来自嗜热热菌的纯化核糖体、延伸因子、tmRNA和SmpB建立了体外系统。制备了一种与多苯丙烯酰trna （Phe）配合的停滞核糖体作为tmRNA的靶标。以smpb依赖的方式观察到从多苯丙烯酰trna （Phe）到丙烯酰tmrna的肽基转移反应。下一个肽基转移到氨基酰基trna特异性地发生在标签肽的假定恢复密码子上，这是通过引入密码子突变来证实的。因此，本研究中开发的体外系统对于研究反翻译的早期步骤是有用的。利用这些体外系统，我们研究了核糖体蛋白S1的功能，该蛋白被认为在翻译中起作用。尽管嗜热T. S1与tmRNA紧密结合，就像大肠杆菌S1一样，但它对反翻译的早期步骤几乎没有影响。
Transfer-messenger RNA (tmRNA) plays a dual role as a tRNA and an mRNA in trans-translation, during which the ribosome replaces mRNA with tmRNA encoding the tag-peptide. These processes have been suggested to involve several tmRNA-binding proteins, including SmpB and ribosomal protein S1. To investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmRNA and SmpB from Thermus thermophilus. A stalled ribosome in complex with polyphenylalanyl-tRNA(Phe) was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNA(Phe) to alanyl-tmRNA was observed in an SmpB-dependent manner. The next peptidyl transfer to aminoacyl-tRNA occurred specifically to the putative resume codon for the tag-peptide, which was confirmed by introducing a mutation in the codon. Thus, the in vitro systems developed in this study are useful to investigate the early steps of trans-translation. Using these in vitro systems, we investigated the function of ribosomal protein S1, which has been believed to play a role in transtranslation. Although T. thermophilus S1 tightly bound to tmRNA, as in the case of Escherichia coli S1, it had little or no effect on the early steps of trans-translation.