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Abstract：
Translation termination is the final step in protein biosynthesis when the synthesized polypeptide is released from the ribosome. Understanding this complex process is important for treatment of many human disorders caused by nonsense mutations in important genes. Here, we present a new method for the analysis of translation termination rate in cell-free systems, CTELS (for C-terminally extended luciferase-based system). This approach was based on a continuously measured luciferase activity during in vitro translation reaction of two reporter mRNA, one of which encodes a C-terminally extended luciferase. This extension occupies a ribosomal polypeptide tunnel and lets the completely synthesized enzyme be active before translation termination occurs, i.e., when it is still on the ribosome. In contrast, luciferase molecule without the extension emits light only after its release. Comparing the translation dynamics of these two reporters allows visualization of a delay corresponding to the translation termination event. We demonstrated applicability of this approach for investigating the effects of cis- and trans-acting components, including small molecule inhibitors and read-through inducing sequences, on the translation termination rate. With CTELS, we systematically assessed negative effects of decreased 3\' UTR length, specifically on termination. We also showed that blasticidin S implements its inhibitory effect on eukaryotic translation system, mostly by affecting elongation, and that an excess of eRF1 termination factor (both the wild-type and a non-catalytic AGQ mutant) can interfere with elongation. Analysis of read-through mechanics with CTELS revealed a transient stalling event at a \"leaky\" stop codon context, which likely defines the basis of nonsense suppression.
摘要：
当合成的多肽从核糖体释放时,翻译终止是蛋白质生物合成的最后步骤。了解这个复杂的过程对于治疗由重要基因中的无义突变引起的许多人类疾病非常重要。这里,我们提出了一种分析无细胞系统中翻译终止率的新方法，CTELS(用于C-末端延伸的基于荧光素酶的系统)。该方法基于在两个报道mRNA的体外翻译反应期间连续测量的荧光素酶活性,其中一个编码C端延伸的荧光素酶。这种延伸占据了核糖体多肽隧道，让完全合成的酶在翻译终止发生之前是有活性的，即，当它还在核糖体上的时候。相比之下,没有延伸的荧光素酶分子仅在其释放后发光。比较这两个报告子的翻译动态允许对应于翻译终止事件的延迟的可视化。我们证明了这种方法对研究顺式和反式作用成分的影响的适用性，包括小分子抑制剂和通读诱导序列，转换终止率。有了CTELS,我们系统地评估了减少3UTR长度的负面影响，特别是在终止。我们还表明杀稻瘟素S对真核翻译系统具有抑制作用，主要是通过影响伸长率，并且过量的eRF1终止因子(野生型和非催化AGQ突变体)可干扰延伸。使用CTELS对通读力学的分析显示，在“泄漏”终止密码子环境下发生了短暂的失速事件，这可能定义了无意义压制的基础。
