Single molecule imaging of the trans-translation entry process via anchoring of the tagged ribosome
Single molecule imaging of the trans-translation entry process via anchoring of the tagged ribosome
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DOI：
10.1093/jb/mvr010
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发表时间：
2011-05-01
影响因子：
2.7
通讯作者：
Ueda, Takuya
中科院分区：
文献类型：
--
作者：
Zhou, Zhan-Ping;Shimizu, Yoshihiro;Ueda, Takuya
反式翻译是真细菌中拯救停滞核糖体的一种质量控制系统，其中涉及转运信使RNA（transfermessagerRNA，tmRNA）和小分子蛋白B（SmpB）等多种成分。然而，这些分子如何与核糖体相互作用仍然难以捉摸。在这里，我们报告的反式翻译过程的单分子分析。我们开发了一种新的标记功能性核糖体的方法，将一个标签蛋白（HaloTag蛋白，由297个氨基酸组成）与30 S核糖体蛋白S2融合，并与特异性配体（HaloTag配体）共价标记，从而实现了对核糖体的稳定、特异性标记。使用Cy 3 HaloTag配体的生物素化衍生物（即生物素-Cy 3-配体）将核糖体锚定到玻璃表面上，并且观察Cy 5-tmRNA/SmpB/EF-Tu三元复合物与锚定的核糖体的实时相互作用作为反式翻译进入的模型。统计分析表明，Cy 5-tmRNA/SmpB/EF-Tu三元复合物与锚定的核糖体结合的二级速率常数为2.6 × 10 ~（6）（1/M/s），tmRNA从核糖体释放前经历多模式途径。本文提出的方法同样适用于一般翻译过程的分析。
Trans-translation is an eubacterial quality control system to rescue the stalled ribosome, in which multiple components such as transfer messenger RNA (tmRNA) and Small protein B (SmpB) are involved. However, how these molecules interact with ribosome remains elusive. Here, we report the single molecule analysis of the trans-translation process. We developed a new method to label the functional ribosome, in which a tag protein (the HaloTag protein of 297 amino acids) was fused to the 30S ribosomal protein S2 and covalently labelled with specific ligand (HaloTag ligand), resulting in the stable and specific labelling of ribosome. Ribosomes were anchored onto the glass surface using biotinylated derivative of the Cy3 HaloTag ligand (i.e. biotin-Cy3-ligand), and real-time interactions of Cy5-tmRNA/SmpB/EF-Tu ternary complexes with anchored ribosomes are observed as a model of the trans-translation entry. Statistical analysis revealed that Cy5-tmRNA/SmpB/EF-Tu ternary complexes bind to the anchored ribosome with the second-order rate constant of 2.6 x 10(6) (1/M/s) and tmRNAs undergo multi-modal pathway before release from ribosome. The methods presented here are also applicable to the analysis for general translation processes.