Termination of protein biosynthesis is an essential step of gene expression, during which a complete functional protein is released from the ribosome. Premature or inefficient termination results in truncated, non-functional or toxic proteins that may cause disease. Indeed, more than 10% of human genetic diseases are caused by nonsense mutations leading to premature termination. Efficient and sensitive approaches are required to study eukaryotic termination mechanisms and to identify potential therapeutics that modulate termination. Canonical radioactivity-based termination assays are complex, report on a short peptide release, and are incompatible with high-throughput screening. Here we describe a robust and simple in vitro assay to study the kinetics of full-protein release. The assay monitors luminescence upon release of nanoluciferase from a mammalian pre-termination complex. The assay can be used to record time-progress curves of protein release in a high-throughput format, making it optimal for studying release kinetics and for high-throughput screening for small molecules that modulate the efficiency of termination.

中文翻译：

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Termi-Luc：一种监测核糖体全蛋白释放的多功能检测方法

蛋白质生物合成的终止是基因表达的重要步骤，在此过程中，完整的功能性蛋白质从核糖体中释放出来。过早或低效终止会导致截短的、无功能的或有毒的蛋白质，从而可能导致疾病。事实上，超过 10% 的人类遗传病是由导致提前终止的无义突变引起的。研究真核细胞终止机制并确定调节终止的潜在疗法需要有效且灵敏的方法。典型的基于放射性的终止测定很复杂，报告短肽释放，并且与高通量筛选不相容。在这里，我们描述了一种强大而简单的体外测定法来研究全蛋白释放的动力学。该测定监测哺乳动物终止前复合物释放纳米荧光素酶时的发光。该测定可用于以高通量形式记录蛋白质释放的时间-进展曲线，使其成为研究释放动力学和调节终止效率的小分子的高通量筛选的最佳选择。