Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, United States.
Howard Hughes Medical Institute, Baltimore, United States.
Elife. 2020 Aug 26;9:e60303. doi: 10.7554/eLife.60303.
Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.
嘌呤霉素是一种酪氨酸-tRNA 类似物，通过标记和释放正在延伸的多肽链，从而阻断翻译。几十年来，嘌呤霉素一直被用于分子生物学研究中，作为一种翻译抑制剂。嘌呤霉素抗体和衍生的嘌呤霉素类似物的开发，使得在批量和单细胞测定中对活性翻译进行定量成为可能。最近，体内嘌呤霉素酰化测定法已成为在细胞中定位翻译核糖体的流行工具。这些测定法通常使用伸长抑制剂来据称抑制从核糖体释放嘌呤霉素标记的新生肽。我们使用各种真核系统中的体外和体内实验证明，即使存在伸长抑制剂，嘌呤霉素酰化的肽也会被释放并从核糖体扩散。嘌呤霉素酰化测定法揭示了亚细胞部位，如细胞核，其中嘌呤霉素酰化的肽在释放后积累，并且不一定与活跃翻译的部位重合。我们的发现敦促在体内解释嘌呤霉素酰化测定法时要谨慎。
