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

Abstract

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.

Importance of diphthamide modified EF2 for translational accuracy and competitive cell growth in yeast

“…Interestingly, a neurological disorder in humans (SCA26) was found to result from a mutation in EF2 that affects a residue close to the diphthamide modified histidine in the 3D structure of EF2 and also induces -1 frameshifting [ 67 ]. In sum, our in vivo experiments together with recent structural and biochemical studies strongly suggest that the role of the diphthamide modification in EF2 is to optimize the efficiency and accuracy of EF2 in ribosomal translation [ 41 , 42 , 68 ].…”

“…Solving the structure at near atomic resolution, the report [ 41 ] concludes that through ensuring reading frame maintenance, diphthamide plays a role in ribosomal translocation during the elongation phase of mRNA translation. Similarly, cryo-EM based structural studies have emerged suggesting that diphthamide may stabilize the interaction of the codon-anticodon duplex and thereby prevent from reverse translocation activity of EF2 [ 42 ]. Interestingly, reducing the interactions of the anticodon mimicry domain IV on EF2 with the codon-anticodon helix by loss of diphthamide [ 42 , 65 , 66 ] could explain the increase in -1 frame-shift errors that research groups including our own have observed in mutants that lack diphthamide modified EF2 or fail to complete diphthamide synthesis [ 19 , 32 , 40 , 61 , 67 ].…”

“…Similarly, cryo-EM based structural studies have emerged suggesting that diphthamide may stabilize the interaction of the codon-anticodon duplex and thereby prevent from reverse translocation activity of EF2 [ 42 ]. Interestingly, reducing the interactions of the anticodon mimicry domain IV on EF2 with the codon-anticodon helix by loss of diphthamide [ 42 , 65 , 66 ] could explain the increase in -1 frame-shift errors that research groups including our own have observed in mutants that lack diphthamide modified EF2 or fail to complete diphthamide synthesis [ 19 , 32 , 40 , 61 , 67 ]. Interestingly, a neurological disorder in humans (SCA26) was found to result from a mutation in EF2 that affects a residue close to the diphthamide modified histidine in the 3D structure of EF2 and also induces -1 frameshifting [ 67 ].…”

“…Based on ribosomal frame-shift assays in yeast, diphthamide has been proposed to support the fidelity of translocation by EF2 during mRNA translation [ 19 , 32 , 40 ]. How this is accomplished, is not entirely clear, and only recently have cryo-EM studies emerged that relate diphthamide modified EF2 in complex with the ribosomal decoding center to accurate mRNA translation [ 41 , 42 ].…”

Complex Evolutionary History of Translation Elongation Factor 2 and Diphthamide Biosynthesis in Archaea and Parabasalids

“…It also seems possible that aEF-2p may compensate for the absence of diphthamide in at least some dph -lacking lineages. Eukaryotic EF-2 has recently been shown to function as a back-translocase ( Susorov et al. 2018 ).…”