Lon protease degrades transfer-messenger RNA-tagged proteins
Lon protease degrades transfer-messenger RNA-tagged proteins
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DOI：
10.1128/jb.00860-07
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发表时间：
2007-09-01
影响因子：
3.2
通讯作者：
Karzai, A. Wali
中科院分区：
文献类型：
--
作者：
Choy, Jennifer S.;Aung, Latt Latt;Karzai, A. Wali
当翻译核糖体不能适当延长或终止时，细菌反式翻译被激活。小分子蛋白B（Smp B）和转移信使RNA（tmRNA）是反式翻译所需的两种已知因子。由ssrA基因编码的tmRNA是一种双功能分子，在反式翻译过程中既作为tRNA又作为mRNA。tmRNA的功能确保停滞的核糖体被拯救，致病缺陷的mRNA被降解，并且不完整的多肽被标记用于靶向蛋白水解。我们目前在体内和体外的证据表明，在降解的tRNA标记的蛋白质的Lon ATP依赖性蛋白酶的直接作用。在内源性蛋白质标记测定中，ton突变体积累了过量的tRNA标记的蛋白质。在用Alzoda-CI-N（设计用于激活反式翻译的不停止mRNA构建体的蛋白产物）进行的报告蛋白标记测定中，Ion突变细胞有效地标记了报告蛋白，但标记的蛋白表现出增加的稳定性。类似地，含有硬编码的C末端tmRNA标签（GFP-SsrA）的绿色荧光蛋白（GFP）构建体在Ion突变细胞中表现出增加的稳定性。最重要的是，与未标记的形式相比，高度纯化的Lon优先降解tRNA标记形式的蛋白质。基于这些结果，我们得出结论，Lon蛋白酶直接参与降解的tRNA标记的蛋白质。
Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, ton mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with lambda-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, Ion mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in Ion mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.