EF4 disengages the peptidyl-tRNA CCA end and facilitates back-translocation on the 70S ribosome

Abstract

EF4 catalyzes tRNA back-translocation through an unknown mechanism. We report cryo-EM structures of Escherichia coli EF4 in post- and pretranslocational ribosomes (Post- and Pre-EF4) at 3.7- and 3.2-Å resolution, respectively. In Post-EF4, peptidyl-tRNA occupies the peptidyl (P) site, but the interaction between its CCA end and the P loop is disrupted. In Pre-EF4, the peptidyl-tRNA assumes a unique position near the aminoacyl (A) site, denoted the A site/EF4 bound (A/4) site, with a large displacement at its acceptor arm. Mutagenesis analyses suggest that a specific region in the EF4 C-terminal domain (CTD) interferes with base-pairing between the peptidyl-tRNA 3'-CCA and the P loop, whereas the EF4 CTD enhances peptidyl-tRNA interaction at the A/4 site. Therefore, EF4 induces back-translocation by disengaging the tRNA's CCA end from the peptidyl transferase center of the translating ribosome.

Structure of the GTP Form of Elongation Factor 4 (EF4) Bound to the Ribosome

“…1C). The unrotated and slightly counterclockwise rotated states of our structures are different from the notable clockwise rotation (30S body rotation by 5°) that was recently reported in the crystal structure of EF4-GDP (14) and the cryo-EM reconstitution of the minor population of EF4-GDPNP (a non-hydrolysable GTP analog) bound to the ribosome (19). On the other hand, the major population of EF4-GDPNP in the recent cryo-EM study and the previous low-resolution reconstitution of the GTP form of EF4 bound to the ribosome with tRNAs both in P and A sites (10) also display unrotated states (10), similar to the major ribosome population in the present structure.…”

“…2B). In contrast, in the recent cryo-EM structure of the E. coli EF4-GDPNP bound to the PRE state ribosomes, Tyr-203 (E. coli numbering, equivalent to Tyr-208 in T. thermophilus LepA) is observed to stack with the Ala-55 residue of the 16S rRNA (19). Similar to the interaction between the EF-G domain III and the ribosome (17), domain III of EF4 in the current structure makes bilateral contacts with both the 30S and 50S subunits, namely, the S12 protein in the 30S shoulder, and the SRL in the 50S subunit, respectively (Fig.…”

“…We were able to build a complete model for EF4, including the regions that are crucial for its function: switch 1 (SW1, residues 38 -60), switch 2 (SW2, residues 81-104), and the unique CTD (residues 490 -610). Although the two switch regions, SW1 and SW2, had been visualized at low resolution based on homology modeling using EF-G and EF-G-2 as templates (10), SW1 was partially traced in EF4-GDPNP bound to the ribosome (19). Therefore, our structure provides a more detailed model for these regions.…”

“…While our manuscript was in preparation, cryo-EM reconstitutions of Escherichia coli EF4 in complex with the PRE and POST state ribosomes were reported (19). The similarities and differences between our structure and these complexes will be discussed.…”

Conserved GTPase LepA (Elongation Factor 4) functions in biogenesis of the 30S subunit of the 70S ribosome

“…Several ribosome-bound LepA structures have been reported (25,(42)(43)(44). In all cases, mRNA and tRNAs were also part of the complex, and the data were often interpreted with the assumption that LepA acts during elongation.…”