Direct measurements of mRNA translation kinetics in living cells

• DOI: 10.1038/s41467-022-29515-x
• Publication Date: 2022-04-06T10:21:16Z
• Authors: Mikhail Metelev; Erik Lundin; Ivan L. Volkov; Arvid H. Gynnå; Johan Elf; Magnus Johansson
• Keywords: 0301 basic medicine; 0303 health sciences; Molekylärbiologi; Science; Q; Biochemistry and Molecular Biology; Biochemistry; Article; Kinetics; 03 medical and health sciences; Protein Biosynthesis; Escherichia coli; RNA, Messenger; Biokemi; Molecular Biology; Ribosomes; Biokemi och molekylärbiologi

Abstract 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 characterize whole processes finding and translating an mRNA, single-molecule tracking techniques. We find that more than 90% both bacterial ribosomal subunits are engaged at any particular time, 30S 50S spend same average time bound revealing re-initiation on poly-cistronic mRNAs not prevalent E. coli . Instead, our results best explained by substantial 70S mRNAs, further corroborated experiments initiation inhibitors. Finally, variety previously described orthogonal ribosomes, altered anti-Shine-Dalgarno sequences, show significant binding endogenous mRNAs.