Elongation factor G initiates translocation through a power stroke

Proc Natl Acad Sci U S A. 2016 Jul 5;113(27):7515-20. doi: 10.1073/pnas.1602668113. Epub 2016 Jun 16.

Abstract

During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G's GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

Keywords: energy landscape; polarized TIRF; ribosome; rotational motion; translocation.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Guanosine Triphosphate / metabolism*
  • Molecular Sequence Data
  • Peptide Elongation Factor G / metabolism*
  • Protein Translocation Systems
  • Ribosomes / metabolism*

Substances

  • Peptide Elongation Factor G
  • Protein Translocation Systems
  • Guanosine Triphosphate