Control of Ribosomal Subunit Rotation by Elongation Factor G

Protein synthesis by the ribosome requires the translocation of transfer RNAs and messenger RNA by one codon after each peptide bond is formed, a reaction that requires ribosomal subunit rotation and is catalyzed by the guanosine triphosphatase (GTPase) elongation factor G (EF-G). We determined 3 angstrom resolution x-ray crystal structures of EF-G complexed with a nonhydrolyzable guanosine 5′-triphosphate (GTP) analog and bound to the Escherichia coli ribosome in different states of ribosomal subunit rotation. The structures reveal that EF-G binding to the ribosome stabilizes switch regions in the GTPase active site, resulting in a compact EF-G conformation that favors an intermediate state of ribosomal subunit rotation. These structures suggest that EF-G controls the translocation reaction by cycles of conformational rigidity and relaxation before and after GTP hydrolysis.

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Elongation factor G initiates translocation through a power stroke

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1602668113 ◽

2016 ◽

Vol 113 (27) ◽

pp. 7515-7520 ◽

Cited By ~ 31

Author(s):

Chunlai Chen ◽

Xiaonan Cui ◽

John F. Beausang ◽

Haibo Zhang ◽

Ian Farrell ◽

...

Keyword(s):

Single Molecule ◽

Messenger Rna ◽

Elongation Factor ◽

Power Stroke ◽

Elongation Factor G ◽

Gtp Hydrolysis ◽

Guanosine Triphosphatase ◽

Prokaryotic Protein ◽

Domain Iii ◽

Factor G

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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Faculty Opinions recommendation of Control of ribosomal subunit rotation by elongation factor G.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718025065.793484584 ◽

2013 ◽

Author(s):

P Shing Ho

Keyword(s):

Ribosomal Subunit ◽

Elongation Factor ◽

Elongation Factor G ◽

Subunit Rotation ◽

Factor G

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Elongation Factor G Bound to the Ribosome in an Intermediate State of Translocation

Science ◽

10.1126/science.1235490 ◽

2013 ◽

Vol 340 (6140) ◽

pp. 1235490 ◽

Cited By ~ 156

Author(s):

David S. Tourigny ◽

Israel S. Fernández ◽

Ann C. Kelley ◽

V. Ramakrishnan

Keyword(s):

Intermediate State ◽

Messenger Rna ◽

Structural Changes ◽

Thermus Thermophilus ◽

Elongation Factor ◽

Chain Elongation ◽

Protein Chain ◽

Elongation Factor G ◽

Guanosine Triphosphatase ◽

Factor G

A key step of translation by the ribosome is translocation, which involves the movement of messenger RNA (mRNA) and transfer RNA (tRNA) with respect to the ribosome. This allows a new round of protein chain elongation by placing the next mRNA codon in the A site of the 30S subunit. Translocation proceeds through an intermediate state in which the acceptor ends of the tRNAs have moved with respect to the 50S subunit but not the 30S subunit, to form hybrid states. The guanosine triphosphatase (GTPase) elongation factor G (EF-G) catalyzes the subsequent movement of mRNA and tRNA with respect to the 30S subunit. Here, we present a crystal structure at 3 angstrom resolution of the Thermus thermophilus ribosome with a tRNA in the hybrid P/E state bound to EF-G with a GTP analog. The structure provides insights into structural changes that facilitate translocation and suggests a common GTPase mechanism for EF-G and elongation factor Tu.

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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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Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0908077106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15702-15707 ◽

Cited By ~ 95

Author(s):

Jingyi Fei ◽

Jonathan E. Bronson ◽

Jake M. Hofman ◽

Rathi L. Srinivas ◽

Chris H. Wiggins ◽

...

Keyword(s):

Dynamic Equilibrium ◽

Ribosomal Subunit ◽

Elongation Factor ◽

Large Ribosomal Subunit ◽

Elongation Factor G ◽

Structural Element ◽

Time Dynamics ◽

Kinetic Mechanisms ◽

Trna Translocation ◽

Factor G

Determining the mechanism by which tRNAs rapidly and precisely transit through the ribosomal A, P, and E sites during translation remains a major goal in the study of protein synthesis. Here, we report the real-time dynamics of the L1 stalk, a structural element of the large ribosomal subunit that is implicated in directing tRNA movements during translation. Within pretranslocation ribosomal complexes, the L1 stalk exists in a dynamic equilibrium between open and closed conformations. Binding of elongation factor G (EF-G) shifts this equilibrium toward the closed conformation through one of at least two distinct kinetic mechanisms, where the identity of the P-site tRNA dictates the kinetic route that is taken. Within posttranslocation complexes, L1 stalk dynamics are dependent on the presence and identity of the E-site tRNA. Collectively, our data demonstrate that EF-G and the L1 stalk allosterically collaborate to direct tRNA translocation from the P to the E sites, and suggest a model for the release of E-site tRNA.

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