Choy Jennifer S, Aung Latt Latt, Karzai A Wali
Department of Biochemistry and Cell Biology, Center for Infectious Diseases, Stony Brook University, Stony Brook, NY 11794, USA.
J Bacteriol. 2007 Sep;189(18):6564-71. doi: 10.1128/JB.00860-07. Epub 2007 Jul 6.
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, lon 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, lon 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 lon 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.
当正在翻译的核糖体无法正常延伸或终止时，细菌反式翻译被激活。小蛋白B（SmpB）和转运信使RNA（tmRNA）是反式翻译所需的且专门用于反式翻译的两个已知因子。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发挥作用。tmRNA的功能确保停滞的核糖体被拯救，导致缺陷的mRNA被降解，不完整的多肽被标记以便进行靶向蛋白水解。我们提供了体内和体外证据，证明Lon ATP依赖性蛋白酶在降解tmRNA标记的蛋白质中起直接作用。在内源蛋白标记试验中，lon突变体积累了过量的tmRNA标记蛋白。在用λ-CI-N进行的报告蛋白标记试验中，λ-CI-N是一种设计用于激活反式翻译的无义mRNA构建体的蛋白产物，lon突变体细胞有效地标记了报告蛋白，但标记的蛋白表现出更高的稳定性。同样，含有硬编码C端tmRNA标签的绿色荧光蛋白（GFP）构建体（GFP-SsrA）在lon突变体细胞中也表现出更高的稳定性。最重要的是，与未标记的形式相比，高度纯化的Lon优先降解tmRNA标记的蛋白形式。基于这些结果，我们得出结论，Lon蛋白酶直接参与tmRNA标记蛋白的降解。
