Real-time tRNA transit on single translating ribosomes at codon resolution

• Published in: Nature (London) Vol. 464; no. 7291; pp. 1012 - 1017
• Main Authors: Puglisi, Joseph D, Uemura, Sotaro, Aitken, Colin Echeverría, Korlach, Jonas, Flusberg, Benjamin A, Turner, Stephen W
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 15.04.2010; Nature Publishing Group
• Subjects: 631/1647/2204; 631/337/574/1789; 631/337/574/1793; Binding Sites; Codon; Codon - genetics; E coli; Escherichia coli; Fluorescence; Genetic translation; Humanities and Social Sciences; Instrumentation; Kinetics; Ligands; Luminescent Measurements; multidisciplinary; Nucleic acids; Optical Tweezers; Properties; Protein biosynthesis; Protein Biosynthesis - genetics; Protein Biosynthesis - physiology; Proteins; Ribosomes; Ribosomes - chemistry; Ribosomes - genetics; Ribosomes - metabolism; RNA, Transfer - genetics; RNA, Transfer - metabolism; Science; Science (multidisciplinary); Time Factors; Translocation; United States
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature08925

Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we use zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically relevant micromolar ligand concentrations. Translation at each codon is monitored by stable binding of transfer RNAs (tRNAs)—labelled with distinct fluorophores—to translating ribosomes, which allows direct detection of the identity of tRNA molecules bound to the ribosome and therefore the underlying messenger RNA (mRNA) sequence. We observe the transit of tRNAs on single translating ribosomes and determine the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA molecule. Our results show that ribosomes are only briefly occupied by two tRNA molecules and that release of deacylated tRNA from the exit (E) site is uncoupled from binding of aminoacyl-tRNA site (A-site) tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation. A single ribosome in action Single-molecule studies allow biological processes to be examined one molecule at a time, as they occur. Here, Uemura et al . have utilized single-molecule approaches with a recently developed technique known as zero-mode waveguide detection, which concentrates reactions in zeptolitre-sized volumes (a zeptolitre is 10 −21 litre) to examine real-time translocation by the ribosome. With this set-up, they are able to follow binding of tRNAs to the ribosome and find that tRNA release from the E and A sites is uncoupled. Single-molecule studies allow biological processes to be examined one molecule at a time, as they occur. Here, zero-mode waveguides have been used to concentrate reactions in zeptolitre-sized volumes, making it possible to study real-time translocation by the ribosome. The binding of transfer RNAs (tRNAs) to the ribosome could be followed; the results show that tRNA release from the exit site is uncoupled from tRNA binding to the aminoacyl-tRNA site.