Elongation factor G initiates translocation through a power stroke.
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.
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6085; Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6083;
Chunlai
C
Chen
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6085; Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6083;
Xiaonan
X
Cui
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6085; Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6083;
John F
JF
Beausang
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323;
Haibo
H
Zhang
Anima Cell Metrology, Inc., Bernardsville, NJ 07924-2270.
Ian
I
Farrell
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323; goldmany@mail.med.upenn.edu cooprman@pobox.upenn.edu.
Barry S
BS
Cooperman
Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6085; Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104-6083; goldmany@mail.med.upenn.edu cooprman@pobox.upenn.edu.
Yale E
YE
Goldman
D000595:Amino Acid Sequence; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008969:Molecular Sequence Data; D020653:Peptide Elongation Factor G / Q000378:metabolism*; D000069284:Protein Translocation Systems; D012270:Ribosomes / Q000378:metabolism*
D020653:Peptide Elongation Factor G; D000069284:Protein Translocation Systems; D006160:Guanosine Triphosphate
energy landscape; polarized TIRF; ribosome; rotational motion; translocation