Rejection of tmRNA·SmpB after GTP hydrolysis by EF-Tu on ribosomes stalled on intact mRNA.
Rejection of tmRNA·SmpB after GTP hydrolysis by EF-Tu on ribosomes stalled on intact mRNA.
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DOI：
10.1261/rna.045773.114
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发表时间：
2014-11
期刊：
影响因子：
--
通讯作者：
Himeno H
中科院分区：
文献类型：
--
作者：
Kurita D;Miller MR;Muto A;Buskirk AR;Himeno H
缺少终止密码子的信使RNA在其3′端捕获核糖体，耗尽可用于蛋白质合成的核糖体库。在细菌中，一个非凡的质量控制系统在一个被称为反式翻译的过程中拯救和消除停滞的核糖体。作为tRNA，转移信使RNA（tmRNA）被氨酰化，由EF-Tu递送到核糖体A位点，并接受新生多肽。然后在tmRNA内的阅读框上恢复翻译，编码靶向新生肽以供蛋白酶降解的短肽标签。反式翻译中一个未解决的问题是tmRNA及其蛋白伴侣SmpB如何优先识别停滞的核糖体而不是积极翻译的核糖体。在这里，我们检查的3′延伸的长度的mRNA的反式翻译的每一步的影响，通过预稳态动力学方法和荧光偏振结合试验。出乎意料的是，EF-Tu激活和GTP水解迅速发生，与mRNA的长度无关，尽管随着mRNA 3′延伸的增加，肽基转移到tmRNA的量减少，并且tmRNA·SmpB与具有长3′延伸的mRNA的核糖体结合不那么紧密。从这些结果中，我们得出结论，tmRNA·SmpB复合物在调节过程中由于下游mRNA和C末端尾对mRNA通道的竞争而解离。调节过程中tmRNA·SmpB复合物的排斥反应，让人联想到经典翻译中核糖体对近同源tRNA的排斥反应。
Messenger RNAs lacking a stop codon trap ribosomes at their 3′ ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3′ extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3′ extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3′ extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.
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影响因子：
10.5
作者：
Gottesman, S;Roche, E;Sauer, RT
通讯作者：
Sauer, RT
影响因子：
10.5
作者：
Herman, C;Thévenet, D;D'Ari, R
通讯作者：
D'Ari, R
影响因子：
14.9
作者：
Hanawa-Suetsugu, K;Takagi, M;Muto, A
通讯作者：
Muto, A
DOI：
10.1073/pnas.91.20.9223
发表时间：
1994-09-27
影响因子：
11.1
作者：
KOMINE, Y;KITABATAKE, M;INOKUCHI, H
通讯作者：
INOKUCHI, H
影响因子：
2.9
作者：
Barends, S;Wower, J;Kraal, B
通讯作者：
Kraal, B