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 and 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 for eukaryotic ribosomes. Here, using a reconstituted mammalian translation system, we show that the 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.
Highlights
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
The process of reverse translocation has been described in detail in bacterial in vitro systems (24 –26, 28); it has not been observed for eukaryotic ribosomes
The A-site of the POST ribosomes contained the UAA stop codon, the E-site was inhabited by the histidine CAU codon, and the P-site was occupied by MVHL–peptidyl-tRNALeu bound to the leucine CUG codon
Summary
The process of reverse translocation has been described in detail in bacterial in vitro systems (24 –26, 28); it has not been observed for eukaryotic ribosomes To address this issue, we assembled eukaryotic ribosomal POST complexes on model mRNA (Fig. S1A) encoding the MVHL tetrapeptide in a reconstituted in vitro mammalian translation system [29, 30]. The POST complexes produced two cDNA fragments: main (127 nucleotides (nt)) and additional (125 nt) (Fig. 1A) The latter appeared because of the presence of a stop codon in the A-site, which is known to adopt a compact conformation leading to mRNA retraction into the A-site [31, 32]. Incubation with eEF2 and deacylated tRNAMet, non-cognate to the E-site, did not cause the toeprint shift (Fig. 1B). Taking into consideration that the shift occurred in a triplet manner and that deacylated tRNA should be cognate to the E-site. similar to reverse translocation in prokaryotes [23, 24], we concluded that we observed reverse translocation of the eukaryotic ribosomes
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