Conformational Changes of Elongation Factor G on the Ribosome during tRNA Translocation

Lin, Jinzhong; Gagnon, Matthieu G.; Bulkley, David; Steitz, Thomas A.

doi:10.1016/j.cell.2014.11.049

Cited by 126 publications

(173 citation statements)

References 61 publications

(94 reference statements)

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“…Perhaps a recent study (11) that found high interdomain mobility of EF-G and its ability to bind the ribosome, containing tRNA in the A-site, in a compact form, gives the answer.…”

Section: Discussionmentioning

confidence: 99%

“…After binding, the protein undergoes conformational changes that bring the second superdomain into the A-site of the SSU (11,12), where it interacts via domain IV with the decoding centre and tRNA-mRNA-complex (12)(13)(14). This interaction disengages the codonanticodon duplex from the decoding centre and allows the SSU head to swivel (12,14).…”

Section: Introductionmentioning

confidence: 99%

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Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Susorov

Zakharov

Shuvalova

et al. 2018

Journal of Biological Chemistry

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During protein synthesis, a ribosome moves along the mRNA template and, using aminoacyl-tRNAs, decodes the template nucleotide triplets to assemble a protein amino acid sequence. This movement is accompanied by shifting of mRNA-tRNA complexes within the ribosome in a process called translocation. In living cells, this process proceeds in a unidirectional manner, bringing the ribosome to the 3′-end of mRNA, and is catalyzed by the GTPase translation elongation factor 2 (EF-G in prokaryotes, eEF2 in eukaryotes). Interestingly, the possibility of spontaneous backward translocation has been shown in vitro for bacterial ribosomes, suggesting a potential reversibility of this reaction. However, this possibility has not yet been tested in eukaryotic cells. Here, using a reconstituted mammalian translation system, we show that eukaryotic elongation factor eEF2 catalyzes ribosomal reverse translocation at one mRNA triplet. We found that this process requires a cognate tRNA in the ribosomal E-site and cannot occur spontaneously without eEF2. The efficiency of this reaction depended on the concentrations of eEF2 and cognate tRNAs and increased in the presence of nonhydrolyzable GTP analogues. Of note, ADP-ribosylation of eEF2 domain IV blocked reverse translocation, suggesting a crucial role of interactions of this domain with the ribosome for the catalysis of the reaction. In summary, our findings indicate that eEF2 is able to induce ribosomal translocation in forward and backward directions highlighting the universal mechanism of tRNA-mRNA movements within the ribosome.

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“…Perhaps a recent study (11) that found high interdomain mobility of EF-G and its ability to bind the ribosome, containing tRNA in the A-site, in a compact form, gives the answer.…”

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Susorov

Zakharov

Shuvalova

et al. 2018

Journal of Biological Chemistry

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show abstract

“…Extensive structural studies (14)(15)(16)(17)(18)(19)(20)(21)(22) of EF-G bound to ribosome have generated a wealth of atomic or near-atomic…”

mentioning

confidence: 99%

Structure of BipA in GTP form bound to the ratcheted ribosome

Kumar

Chen

Ero

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3′,5′-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation.

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“…These alterations in the SRL result in a complete block of translation, which is thought to be the consequence of an impaired activation/binding of trGTPases (17). Recent crystal structures of EF-Tu (12), or EF-G (9,(18)(19)(20)(21) bound to the ribosome stimulated considerations about the mechanism of ribosome-triggered GTP hydrolysis. Based on the close proximity of the phosphate oxygen at position A2662 of the SRL to the supposedly catalytic histidine of EF-Tu (His84) or EF-G (His87) (Fig.…”

mentioning

confidence: 99%

Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

Koch

Flür

Kreutz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

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Conformational Changes of Elongation Factor G on the Ribosome during tRNA Translocation

Cited by 126 publications

References 61 publications

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

Structure of BipA in GTP form bound to the ratcheted ribosome

Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

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