Insights into the molecular determinants of EF-G catalyzed translocation. Academic Article uri icon

Overview

MeSH

  • Binding Sites
  • Fluorescence Resonance Energy Transfer
  • Histones
  • Microscopy, Fluorescence
  • Models, Molecular
  • Mutation
  • Nucleic Acid Conformation
  • RNA Transport
  • Ribosomes

MeSH Major

  • Peptide Elongation Factor G
  • RNA, Messenger
  • RNA, Transfer

abstract

  • Translocation, the directional movement of transfer RNA (tRNA) and messenger RNA (mRNA) substrates on the ribosome during protein synthesis, is regulated by dynamic processes intrinsic to the translating particle. Using single-molecule fluorescence resonance energy transfer (smFRET) imaging, in combination with site-directed mutagenesis of the ribosome and tRNA substrates, we show that peptidyl-tRNA within the aminoacyl site of the bacterial pretranslocation complex can adopt distinct hybrid tRNA configurations resulting from uncoupled motions of the 3'-CCA terminus and the tRNA body. As expected for an on-path translocation intermediate, the hybrid configuration where both the 3'-CCA end and body of peptidyl-tRNA have moved in the direction of translocation exhibits dramatically enhanced puromycin reactivity, an increase in the rate at which EF-G engages the ribosome, and accelerated rates of translocation. These findings provide compelling evidence that the substrate for EF-G catalyzed translocation is an intermediate wherein the bodies of both tRNA substrates adopt hybrid positions within the translating ribosome.

publication date

  • December 2011

has subject area

  • Binding Sites
  • Fluorescence Resonance Energy Transfer
  • Histones
  • Microscopy, Fluorescence
  • Models, Molecular
  • Mutation
  • Nucleic Acid Conformation
  • Peptide Elongation Factor G
  • RNA Transport
  • RNA, Messenger
  • RNA, Transfer
  • Ribosomes

Research

keywords

  • Journal Article

Identity

Language

  • eng

PubMed Central ID

  • PMC3222131

Digital Object Identifier (DOI)

  • 10.1261/rna.029033.111

PubMed ID

  • 22033333

Additional Document Info

start page

  • 2189

end page

  • 2200

volume

  • 17

number

  • 12