Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation

Nat Struct Mol Biol. 2016 Apr;23(4):333-41. doi: 10.1038/nsmb.3177. Epub 2016 Feb 29.


Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Escherichia coli / chemistry
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / analysis
  • Escherichia coli Proteins / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Guanosine Diphosphate / analysis
  • Guanosine Diphosphate / metabolism
  • Models, Molecular
  • Peptide Elongation Factor G / analysis
  • Peptide Elongation Factor G / metabolism*
  • Protein Transport
  • RNA, Bacterial / analysis
  • RNA, Bacterial / metabolism*
  • RNA, Transfer, Amino Acyl / analysis
  • RNA, Transfer, Amino Acyl / metabolism*
  • Ribosomes / chemistry
  • Ribosomes / metabolism*


  • Escherichia coli Proteins
  • Peptide Elongation Factor G
  • RNA, Bacterial
  • RNA, Transfer, Amino Acyl
  • tRNA, peptidyl-
  • Guanosine Diphosphate