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

Sotaro Uemura, Colin Echeverría Aitken, Jonas Korlach, Benjamin A. Flusberg, Stephen W. Turner and Joseph D. Puglisi ()
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Sotaro Uemura: Stanford University School of Medicine, Stanford, California 94305-5126, USA
Colin Echeverría Aitken: Stanford University School of Medicine, Stanford, California 94305-5126, USA
Jonas Korlach: Pacific Biosciences, Inc., 1505 Adams Drive, Menlo Park, California 94025-1451, USA
Benjamin A. Flusberg: Pacific Biosciences, Inc., 1505 Adams Drive, Menlo Park, California 94025-1451, USA
Stephen W. Turner: Pacific Biosciences, Inc., 1505 Adams Drive, Menlo Park, California 94025-1451, USA
Joseph D. Puglisi: Stanford University School of Medicine, Stanford, California 94305-5126, USA

Nature, 2010, vol. 464, issue 7291, 1012-1017

Abstract: 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.

Date: 2010
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DOI: 10.1038/nature08925

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