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

Authors :
Uemura S
Aitken CE
Korlach J
Flusberg BA
Turner SW
Puglisi JD
Source :
Nature [Nature] 2010 Apr 15; Vol. 464 (7291), pp. 1012-7.
Publication Year :
2010

Abstract

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.

Details

Language :
English
ISSN :
1476-4687
Volume :
464
Issue :
7291
Database :
MEDLINE
Journal :
Nature
Publication Type :
Academic Journal
Accession number :
20393556
Full Text :
https://doi.org/10.1038/nature08925