Nature pap

Sotaro Uemura, Colin Echeverría Aitken, Jonas Korlach, Benjamin A. Flusberg, Stephen W. Turner & Joseph D. Puglisi. Real-time tRNA transit on single translating ribosomes at codon resolution // Nature 464, 1012-1017 (15 April 2010) doi:10.1038/nature08925

http://www.nature.com/nature/journal/v464/n7291/abs/nature08925.html

Article

Nature 464, 1012-1017 (15 April 2010) | doi:10.1038/nature08925; Received 22 January 2010; Accepted 18 February 2010

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

Sotaro Uemura1,2, Colin Echeverría Aitken1,3,5, Jonas Korlach4, Benjamin A. Flusberg4, Stephen W. Turner4 & Joseph D. Puglisi1,3

  1. Department of Structural Biology, Stanford University School of Medicine, Stanford, California 94305-5126, USA
  2. Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan
  3. Biophysics Program, Stanford University School of Medicine, Stanford, California 94305-5126, USA
  4. Pacific Biosciences, Inc., 1505 Adams Drive, Menlo Park, California 94025-1451, USA
  5. 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.

Correspondence to: Joseph D. Puglisi1,3 Correspondence and requests for materials should be addressed to J.D.P. (Email: puglisi@stanford.edu).

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.

Real-time tRNA transit on single translating ribosomes at codon resolution p1012

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.

Sotaro Uemura, Colin Echeverría Aitken, Jonas Korlach, Benjamin A. Flusberg, Stephen W. Turner & Joseph D. Puglisi

doi:10.1038/nature08925

Abstract | Full Text | PDF (939K) | Supplementary information

See also: Editor's summary | News and Views by Brakmann

News and Views

Nature 464, 987-988 (15 April 2010) | doi:10.1038/464987a; Published online 14 April 2010

Single-molecule analysis: A ribosome in action

Susanne Brakmann1

The manufacture of proteins by ribosomes involves complex interactions of diverse nucleic-acid and protein ligands. Single-molecule studies allow us, for the first time, to follow the synthesis of full-length proteins in real time.

Protein synthesis involves a complex interplay of various cellular components. Ribosomes are the cell's protein-production factories, and interact with messenger RNA (the template), amino-acylated transfer RNAs (which act as adaptors between mRNA and amino-acid residues) and diverse co-factors (for the initiation of synthesis, elongation of the nascent chain and release of the mature polypeptide).

  1. Susanne Brakmann is in the Department of Chemical Biology, Technische Universität Dortmund, D-44227 Dortmund, Germany.
    1. Email: susanne.brakmann@tu-dortmund.de

Abstract

Editor's Summary

15 April 2010

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.

NEWS AND VIEWS

:Single-molecule analysis: A ribosome in action

The manufacture of proteins by ribosomes involves complex interactions of diverse nucleic-acid and protein ligands. Single-molecule studies allow us, for the first time, to follow the synthesis of full-length proteins in real time.

Susanne Brakmann

doi:10.1038/464987a

Full Text | PDF (507K)

ARTICLE

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

Sotaro Uemura, Colin Echeverría Aitken, Jonas Korlach, Benjamin A. Flusberg, Stephen W. Turner & Joseph D. Puglisi

doi:10.1038/nature08925

Abstract | Full Text | PDF (939K) | Supplementary information