Conformational Changes of the Small Ribosomal Subunit During Elongation Factor G-dependent tRNA–mRNA Translocation

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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Effect of propan-2-ol on enzymic and structural properties of elongation factor G

Biochemical Journal ◽

10.1042/bj2610725 ◽

1989 ◽

Vol 261 (3) ◽

pp. 725-731 ◽

Cited By ~ 5

Author(s):

M Masullo ◽

G Parlato ◽

E De Vendittis ◽

V Bocchini

Keyword(s):

Conformational Changes ◽

Structural Changes ◽

Elongation Factor ◽

Elongation Factor G ◽

Fluorescence Measurements ◽

Different Temperatures ◽

Number Of Binding Sites ◽

Kinetics Of ◽

Factor G

Elongation factor G (EF-G) can support a GTPase activity in vitro even in the absence of ribosomes when propan-2-ol is present [GTPasep; De Vendittis, Masullo & Bocchini (1986) J. Biol. Chem. 261, 4445-4450]. In the present work the GTPasep activity of EF-G was further studied by investigating (i) the effect of ionic environment on GTPasep and (ii) the influence of propan-2-ol on the molecular structure of EF-G as determined by fluorescence and c.d. measurements. In the presence of 1-300 mM univalent cations (M+) alone, no detectable GTPasep activity was measured; however, in the presence of 1 mM-Mg2+ a considerable stimulation was observed at 40 mM-Li+ or 75 mM-NH4+. Among bivalent cations (M2+), 1 mM-Sr2+, 2-5 mM-Ca2+ and 1 mM-Ba2+ were the most effective, but, in the presence of 75 mM-NH4+, Mg2+ and Mn2+ became the most efficient, whereas the stimulation by other M2+ species was considerably decreased. C.d. measurements showed that the alcohol increased the mean molar residue ellipticity of EF-G at 285 nm, but not at 220 nm. As estimated from fluorescence measurements, in the presence of 20% (v/v) propan-2-ol the value of the dissociation constant of the complex formed between EF-G and 8-anilino-1-naphthalene-sulphonate decreased from 8 to 5 microM; similarly, the number of binding sites on EF-G for the fluorescent probe decreased from 13 to 6. Finally, the alcohol enhanced the quenching of the intrinsic fluorescence of EF-G caused by either acrylamide or KI. The data support the hypothesis that propan-2-ol induces moderate conformational changes of EF-G that make the catalytic centre accessible to the substrate even in the absence of ribosomes. Kinetics of GTPasep studied at different temperatures did not reveal additional structural changes of EF-G occurring with time or temperature.

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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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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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