Metelev Mikhail, Lundin Erik, Volkov Ivan L, Gynnå Arvid H, Elf Johan, Johansson Magnus
Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden.
Nat Commun. 2022 Apr 6;13(1):1852. doi: 10.1038/s41467-022-29515-x.
Ribosome mediated mRNA translation is central to life. The cycle of translation, however, has been characterized mostly using reconstituted systems, with only few techniques applicable for studies in the living cell. Here we describe a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding and translating an mRNA, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in translation at any particular time, and that the 30S and 50S ribosomal subunits spend the same average time bound to an mRNA, revealing that 30S re-initiation on poly-cistronic mRNAs is not prevalent in E. coli. Instead, our results are best explained by substantial 70S re-initiation of translation of poly-cistronic mRNAs, which is further corroborated by experiments with translation initiation inhibitors. Finally, we find that a variety of previously described orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs.
核糖体介导的mRNA翻译是生命的核心。然而，翻译循环大多是使用重组系统来表征的，仅有少数技术适用于活细胞研究。在此，我们描述了一种活细胞核糖体标记方法，该方法使我们能够利用单分子追踪技术来表征寻找和翻译mRNA的整个过程。我们发现，在任何特定时间，超过90%的细菌核糖体亚基都参与了翻译，并且30S和50S核糖体亚基与mRNA结合的平均时间相同，这表明多顺反子mRNA上的30S重新起始在大肠杆菌中并不普遍。相反，我们的结果最好由多顺反子mRNA翻译的大量70S重新起始来解释，这一点通过翻译起始抑制剂实验得到了进一步证实。最后，我们发现，各种先前描述的具有改变的抗Shine-Dalgarno序列的正交核糖体，对内源mRNA显示出显著的结合。
