Department of Cell and Molecular Biology, Uppsala University, Sweden.
Department of Cell and Molecular Biology, Uppsala University, Sweden.
J Mol Biol. 2021 May 14;433(10):166942. doi: 10.1016/j.jmb.2021.166942. Epub 2021 Mar 18.
Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNA on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNA. By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNA, underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.
大环内酯类抗生素，如红霉素，与细菌核糖体的新生肽出口隧道（NPET）结合，并根据新生肽序列调节蛋白质合成。虽然体外生化和结构方法在剖析和解释大环内酯类药物诱导的肽基-tRNA 脱落和核糖体停滞的分子细节方面发挥了重要作用，但这些药物对活细菌细胞内正在进行的蛋白质合成的动态影响还远未得到探索。在本研究中，我们使用荧光标记 tRNA 的单颗粒追踪技术，直接在活大肠杆菌细胞内研究红霉素存在时 mRNA 翻译的动力学。在红霉素处理的细胞中，我们发现延伸 tRNA 在核糖体上的停留时间显著延长，但发生的频率要低得多。相比之下，该药物几乎不影响起始 tRNA 的核糖体结合事件。通过过表达特定的短肽，我们进一步发现 tRNA 的核糖体结合动力学具有特定的上下文，突出了红霉素对细菌细胞中蛋白质合成的影响的复杂性。
