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.

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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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Control of Ribosomal Subunit Rotation by Elongation Factor G

Science ◽

10.1126/science.1235970 ◽

2013 ◽

Vol 340 (6140) ◽

pp. 1235970 ◽

Cited By ~ 115

Author(s):

Arto Pulk ◽

Jamie H. D. Cate

Keyword(s):

Messenger Rna ◽

Ribosomal Subunit ◽

Elongation Factor ◽

Peptide Bond ◽

Elongation Factor G ◽

Before And After ◽

Switch Regions ◽

Subunit Rotation ◽

Guanosine Triphosphatase ◽

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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Domain III of Elongation Factor G fromThermus thermophilusIs Essential for Induction of GTP Hydrolysis on the Ribosome

Journal of Biological Chemistry ◽

10.1074/jbc.m002656200 ◽

2000 ◽

Vol 275 (46) ◽

pp. 35820-35824 ◽

Cited By ~ 20

Author(s):

Kirill A. Martemyanov ◽

Anatoly T. Gudkov

Keyword(s):

Elongation Factor ◽

Elongation Factor G ◽

Gtp Hydrolysis ◽

Domain Iii ◽

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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Crystallographic studies of elongation factor G

Biochemistry and Cell Biology ◽

10.1139/o95-130 ◽

1995 ◽

Vol 73 (11-12) ◽

pp. 1209-1216 ◽

Cited By ~ 17

Author(s):

Anders Liljas ◽

Arnthor Ævarsson ◽

Salam Al-Karadaghi ◽

Maria Garber ◽

Julia Zheltonosova ◽

...

Keyword(s):

Crystal Structures ◽

Conformational Change ◽

Conformational Changes ◽

Elongation Factor ◽

Elongation Factors ◽

Elongation Factor G ◽

Conformational States ◽

Gtp Hydrolysis ◽

Large Conformational Change ◽

Factor G

The elongation factors G (EF-G) and Tu (EF-Tu) go through a number of conformation states in their functional cycles. Since they both are GTPases, have similar G domains and domains II, and have similar interactions with the nucleotides, then GTP hydrolysis must occur in similar ways. The crystal structures of two conformational states are known for EF-G and three are known for EF-Tu. The conformations of EF-G∙GDP and EF-Tu∙GTP are closely related. EF-Tu goes through a large conformational change upon GTP cleavage. This conformational change is to a large extent due to an altered interaction between the G domain and domains II and III. A number of kirromycin-resistant mutations are situated at the interface between domains I and III. The interface between the G domain and domain V in EF-G corresponds with this dynamic interface in EF-Tu. The contact area in EF-G is small and dominated by interactions between charged amino acids, which are part of a system that is observed to undergo conformational changes. Furthermore, a number of fusidic acid resistant mutants have been identified in this area. All of this evidence makes it likely that EF-G undergoes a large conformational change in its functional cycle. If the structures and conformational states of the elongation factors are related to a scheme in which the ribosome oscillates between two conformations, the pretranslocational and posttranslocational states, a model is arrived at in which EF-Tu drives the reaction in one direction and EF-G in the opposite. This may lead to the consequence that the GTP state of one factor is similar to the GDP state of the other. At the GTP hydrolysis state, the structures of the factors will be close to superimposable.Key words: elongation factor G, elongation factor Tu, crystal structures, conformational changes, ribosomal conformation.

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