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A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation

Abstract

EF4 (LepA) is an almost universally conserved translational GTPase in eubacteria. It seems to be essential under environmental stress conditions and has previously been shown to back-translocate the tRNAs on the ribosome, thereby reverting the canonical translocation reaction. In the current work, EF4 was directly visualized in the process of back-translocating tRNAs by single-particle cryo-EM. Using flexible fitting methods, we built a model of ribosome-bound EF4 based on the cryo-EM map and a recently published unbound EF4 X-ray structure. The cryo-EM map establishes EF4 as a noncanonical elongation factor that interacts not only with the elongating ribosome, but also with the back-translocated tRNA in the A-site region, which is present in a previously unseen, intermediate state and deviates markedly from the position of a canonical A-tRNA. Our results, therefore, provide insight into the underlying structural principles governing back-translocation.

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Figure 1: The cryo-EM reconstruction of the 70S–EF4 complex.
Figure 2: Molecular model of ribosome-bound EF4.
Figure 3: The A/L-tRNA.

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Acknowledgements

We thank M. Pech for his help and discussions, and A. Sali and W. Chiu for their support in the development of the cryo-EM density fitting methods. The present work was supported by grants from the Volkswagen Stiftung (C.M.T.S.), the Deutsche Forschungsgemeinschaft (DFG; SF8740 and SP 1130/2-1 to C.M.T.S. and K.H.N.), by the European Union 3D-EM Network of Excellence and by the European Union and Senatsverwaltung für Wissenschaft, Forschung und Kultur Berlin (UltraStructureNetwork, Anwenderzentrum). S.R.C. was supported with a grant from the Alexander von Humboldt Stiftung. M.T. is supported by a Medical Research Council Career Development Award (G0600084).

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Correspondence to Christian M T Spahn.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–4, Supplementary Results and Supplementary Methods. (PDF 6212 kb)

Supplementary Movie

Resolution of the switch 1 region in the cryo-EM reconstruction. The switch 1 region of EF4 (red) has been modeled according to the corresponding region seen in the X-ray structure of EF-G-2 (PDB 1WDT). The cryo-EM density map is illustrated as a wire mesh and has been rendered at two thresholds. The switch 1 region (as modeled on the X-ray structure) does not directly correspond to the density and therefore it is presumed that it undergoes a conformational change on the ribosome. The cryo-EM density suggests that this conformational change would lead to the distal end of the loop forming an interaction near helix 14 (orange) of the 16S rRNA. The helical region in the distal end of the switch region appears to interact with domain III (of EF4; cyan) and H95 (purple) of the 23S rRNA. The movie was prepared with PyMOL. (MOV 4282 kb)

Supplementary Movie 2

A 360 degree rotation around the 3' CCA end of the A/L-tRNA (blue). The cryo-EM density is shown as a wire mesh and rendered at the relatively high 3 sigma threshold such that the various elements remain well defined although somewhat fragmented. The gap between the H71 and 92 (the H71/92 corridor) is clearly occupied by density, and it is within this density that the 3' CCA end of the A/L-tRNA has been modeled. (MOV 1675 kb)

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Connell, S., Topf, M., Qin, Y. et al. A new tRNA intermediate revealed on the ribosome during EF4-mediated back-translocation. Nat Struct Mol Biol 15, 910–915 (2008). https://doi.org/10.1038/nsmb.1469

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