Viruses use internal ribosome entry sites (IRES) to hijack host ribosomes and promote cap-independent translation. Although they are well-studied in bulk, the dynamics of IRES-mediated translation remain unexplored at the single-molecule level. Here, we developed a bicistronic biosensor encoding distinct repeat epitopes in two open reading frames (ORFs), one translated from the 5′ cap, and the other from the encephalomyocarditis virus IRES. When combined with a pair of complementary probes that bind the epitopes cotranslationally, the biosensor lights up in different colors depending on which ORF is translated. Using the sensor together with single-molecule tracking and computational modeling, we measured the kinetics of cap-dependent versus IRES-mediated translation in living human cells. We show that bursts of IRES translation are shorter and rarer than bursts of cap translation, although the situation reverses upon stress. Collectively, our data support a model for translational regulation primarily driven by transitions between translationally active and inactive RNA states.

病毒使用内部核糖体进入位点 (IRES) 来劫持宿主核糖体并促进与帽无关的翻译。尽管对它们进行了大量研究，但 IRES 介导的翻译动力学在单分子水平上仍未得到探索。在这里，我们开发了一种双顺反子生物传感器，在两个开放阅读框 (ORF) 中编码不同的重复表位，一个从 5' 帽翻译，另一个来自脑心肌炎病毒 IRES。当与一对共翻译结合表位的互补探针结合时，生物传感器会根据翻译的 ORF 以不同的颜色点亮。将传感器与单分子跟踪和计算建模结合使用，我们测量了活人细胞中帽依赖性与 IRES 介导的翻译动力学。我们表明，尽管情况在压力下逆转，但 IRES 翻译的爆发比帽翻译的爆发更短、更罕见。总的来说，我们的数据支持主要由翻译活性和非活性 RNA 状态之间的转换驱动的翻译调节模型。