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

Author

Listed:
  • Sotaro Uemura

    (Stanford University School of Medicine, Stanford, California 94305-5126, USA
    Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan)

  • Colin Echeverría Aitken

    (Stanford University School of Medicine, Stanford, California 94305-5126, USA
    Biophysics Program, Stanford University School of Medicine, Stanford, California 94305-5126, USA
    Present address: Department of Biophysics and Biophysical Chemistry, John Hopkins University School of Medicine, 725 N. Wolfe Street, WBSB 713, Baltimore, Maryland 21205-2185, 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
    Biophysics Program, Stanford University School of Medicine, Stanford, California 94305-5126, USA)

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.

Suggested Citation

  • Sotaro Uemura & Colin Echeverría Aitken & Jonas Korlach & Benjamin A. Flusberg & Stephen W. Turner & Joseph D. Puglisi, 2010. "Real-time tRNA transit on single translating ribosomes at codon resolution," Nature, Nature, vol. 464(7291), pages 1012-1017, April.
    • Handle: RePEc:nat:nature:v:464:y:2010:i:7291:d:10.1038_nature08925
    DOI: 10.1038/nature08925
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