SmpB functions in various steps of trans-translation
SmpB functions in various steps of trans-translation
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DOI：
10.1093/nar/30.7.1620
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发表时间：
2002-04-01
影响因子：
14.9
通讯作者：
Muto, A
中科院分区：
文献类型：
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作者：
Hanawa-Suetsugu, K;Takagi, M;Muto, A
tmRNA具有tRNA和mRNA的双重功能，以促进反式翻译，其中核糖体可以在截断的mRNA和tmRNA的标签序列之间切换翻译。SmpB是一种tmRNA结合蛋白，已被确定为体内反翻译所必需的。为了进一步研究SmpB的功能，我们从一株大肠杆菌中提取S30片段，将SmpB和tmRNA的一组基因从基因组中删除，制备了具有反翻译活性的his标记的SmpB。当将纯化的his标记的SmpB与tmRNA一起外源添加时，SmpB缺失的S30片段能够促进体外聚(U)依赖性标签肽的合成，尽管体外聚(U)依赖性聚（Phe）合成不需要SmpB。我们还发现，SmpB的消耗导致细胞中tmRNA水平的降低。此外，在体外实验中，SmpB显著增强了alanyl-tRNA合成酶对tmRNA的氨基化作用。SmpB的氨基酰化增强、SmpB与tmRNA的结合以及SmpB缺失对细胞中tmRNA表达水平的影响都受到trna样结构域的一些突变的影响，这些突变导致核糖体结合缺陷导致反翻译缺陷。这些结果表明，SmpB通过与tmRNA的trna样结构域结合，发挥了多种作用：挽救tmRNA分子在细胞内的降解，增强tmRNA的氨基酰化，介导tmRNA与核糖体的结合。
tmRNA has a dual function as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA's tag sequence. SmpB is a tmRNA binding protein that has been identified to be essential for trans-translation in vivo. To further study the function of SmpB, an S30 fraction from an Escherichia coli strain, in which the set of genes for SmpB and tmRNA has been deleted from the genome, and His-tagged SmpB active in trans-translation were prepared. The SmpB-depleted S30 fraction had an ability to facilitate poly(U)-dependent tag-peptide synthesis in vitro when purified His-tagged SmpB was exogenously added together with tmRNA, although SmpB was not required for in vitro poly(U)-dependent poly(Phe) synthesis. It was also found that depletion of SmpB leads to a decrease in the level of tmRNA in the cell. In addition, SmpB considerably enhanced the aminoacylation of tmRNA by alanyl-tRNA synthetase in vitro. The aminoacylation enhancement by SmpB, the binding of SmpB to tmRNA and the effect of depletion of SmpB on the expression level of tmRNA in the cell were all affected by some mutations in the tRNA-like domain which cause a defect in ribosome binding leading to a trans-translation deficiency. These results demonstrate that, via binding to the tRNA-like domain of tmRNA, SmpB plays various roles: rescuing the tmRNA molecule from degradation in the cell, enhancing the aminoacylation of tmRNA and mediating the binding of tmRNA to ribosome.