A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation
A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation
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DOI：
10.1261/rna.604907
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发表时间：
2007-10-01
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：
Konno, Takayuki;Kurita, Daisuke;Himeno, Hyouta
在反翻译中，具有tRNA和mRNA双重功能的传递信使RNA （transfer-messenger RNA, tmRNA）在SmpB的帮助下缓解核糖体上停滞的翻译。在此，我们建立了一个体外系统，利用大肠杆菌翻译和反翻译因子来评价反翻译的两个步骤，肽基从肽基trna向丙烯基tmRNA的转移和tmRNA上resume密码子的翻译。利用该系统，研究了标签编码区上游的几个突变对tmRNA的影响。这些突变影响了恢复密码子的翻译，而不是肽基转移，其中一个突变A84U/U85G导致了恢复密码子的-1移位。我们还发现U-85不受SmpB的化学修饰。在A84U/U85G突变体中，保护碱基从85转移到84。另一个突变，A86U，导致恢复密码子移动+1，将保护碱基从85转移到86。85时的保护作用被对SmpB结合至关重要的trna样结构域的突变所抑制。这些结果表明SmpB可以桥接tmRNA的两个独立结构域，以确定标签翻译的初始密码子。截断非结构化c端尾部的突变体SmpB未能促进肽基转移，尽管它仍然保护U85免受化学修饰。
In trans-translation, transfer-messenger RNA (tmRNA), possessing a dual function as a tRNA and an mRNA, relieves a stalled translation on the ribosome with the help of SmpB. Here, we established an in vitro system using Escherichia coli translation and trans-translation factors to evaluate two steps of trans-translation, peptidyl transfer from peptidyl-tRNA to alanyl-tmRNA and translation of the resume codon on tmRNA. Using this system, the effects of several mutations upstream of the tag-encoding region on tmRNA were examined. These mutations affected translation of the resume codon rather than peptidyl transfer, and one of them, A84U/U85G, caused a shift of the resume codon by -1. We also found that U-85 is protected from chemical modification by SmpB. In the A84U/U85G mutant, the base of protection was shifted from 85 to 84. Another mutation, A86U, which caused a shift of the resume codon by +1, shifted the base of protection from 85 to 86. The protection at 85 was suppressed by a mutation in the tRNA-like domain critical to SmpB binding. These results suggest that SmpB serves to bridge two separate domains of tmRNA to determine the initial codon for tag-translation. A mutant SmpB with a truncation of the unstructured C-terminal tail failed to promote peptidyl transfer, although it still protected U85 from chemical modification.