Purification and Characterization of an Inhibitor of Elongation Factor G-dependent Guanosine Triphosphatase Reaction of Ribosomes from Ribosome Wash of Escherichia coli Q13

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.

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Activation of GTP hydrolysis in mRNA-tRNA translocation by elongation factor G

Science Advances ◽

10.1126/sciadv.1500169 ◽

2015 ◽

Vol 1 (4) ◽

pp. e1500169 ◽

Cited By ~ 29

Author(s):

Wen Li ◽

Zheng Liu ◽

Ravi Kiran Koripella ◽

Robert Langlois ◽

Suparna Sanyal ◽

...

Keyword(s):

Elongation Factor ◽

Elongation Factor G ◽

Direct Role ◽

Gtp Hydrolysis ◽

Ribosomal Subunits ◽

Trna Translocation ◽

Gtpase Activation ◽

Guanosine Triphosphatase ◽

Factor G

During protein synthesis, elongation of the polypeptide chain by each amino acid is followed by a translocation step in which mRNA and transfer RNA (tRNA) are advanced by one codon. This crucial step is catalyzed by elongation factor G (EF-G), a guanosine triphosphatase (GTPase), and accompanied by a rotation between the two ribosomal subunits. A mutant of EF-G, H91A, renders the factor impaired in guanosine triphosphate (GTP) hydrolysis and thereby stabilizes it on the ribosome. We use cryogenic electron microscopy (cryo-EM) at near-atomic resolution to investigate two complexes formed by EF-G H91A in its GTP state with the ribosome, distinguished by the presence or absence of the intersubunit rotation. Comparison of these two structures argues in favor of a direct role of the conserved histidine in the switch II loop of EF-G in GTPase activation, and explains why GTP hydrolysis cannot proceed with EF-G bound to the unrotated form of the ribosome.

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