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Initiation factor eIF2γ promotes eIF2–GTP–Met-tRNAiMet ternary complex binding to the 40S ribosome

Nature Structural & Molecular Biology volume 18, pages 1227–1234 (2011)Cite this article

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Abstract

In contrast to prokaryotic elongation factor EF-Tu, which delivers aminoacyl-tRNAs to the ribosomal A-site, eukaryotic initiation factor eIF2 binds methionyl initiator transfer RNA (Met-tRNAiMet) to the P-site of the 40S ribosomal subunit. The results of directed hydroxyl radical probing experiments to map binding of Saccharomyces cerevisiae eIF2 on the ribosome and on Met-tRNAiMet revealed that eIF2γ primarily contacts the acceptor stem of Met-tRNAiMet and identified a key binding interface between domain III of eIF2γ and 18S rRNA helix h44 on the 40S subunit. Whereas the analogous domain III of EF-Tu contacts the T stem of tRNAs, biochemical analyses demonstrated that eIF2γ domain III is important for ribosome, not Met-tRNAiMet. Thus, despite their structural similarity, eIF2 and EF-Tu bind tRNAs in substantially different manners, and we propose that the tRNA-binding domain III of EF-Tu has acquired a new ribosome-binding function in eIF2γ.

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Figure 1: Structures of EF-Tu ternary complex and aIF2γ ternary complex model.
Figure 2: Construction and analysis of eIF2 cysteine mutants.
Figure 3: Directed hydroxyl radical cleavage of Met-[32P]tRNAiMet by Fe(II)-BABE–derivatized eIF2 in 48S complexes.
Figure 4: Directed hydroxyl radical cleavage of 18S rRNA by Fe(II)-BABE–derivatized eIF2 in 48S complexes.
Figure 5: eIF2γ binding site for 3′ end of Met-tRNAiMet.
Figure 6: eIF2γ domain III is involved in 40S binding.
Figure 7: 43S complex model.

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Acknowledgements

We thank A. Hinnebusch, R. Green and our colleagues in the Dever, Lorsch and Hinnebusch laboratories for advice and helpful discussions. We thank J. Fringer (US National Institutes of Health), D. Eyler, S. He, H. Zaher (all Johns Hopkins University) and O. Uhlenbeck (Northwestern University) for protocols and reagents. This work was supported in part by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, US National Institutes of Health (T.E.D.) and by grant GM62128 from the NIH (J.R.L.).

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  1. Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

    Byung-Sik Shin, Joo-Ran Kim, Jinsheng Dong & Thomas E Dever

  2. Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

    Sarah E Walker & Jon R Lorsch

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Contributions

B.-S.S. and J.-R.K. carried out the mutagenesis and protein purification; B.-S.S. conducted the hydroxyl radical mapping experiments, biochemical analyses and model building; S.E.W. did the toe-printing assay and J.D. provided reagents. The manuscript was prepared by B.-S.S., S.E.W., J.R.L. and T.E.D.

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Correspondence to Thomas E Dever.

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Shin, BS., Kim, JR., Walker, S. et al. Initiation factor eIF2γ promotes eIF2–GTP–Met-tRNAiMet ternary complex binding to the 40S ribosome. Nat Struct Mol Biol 18, 1227–1234 (2011). https://doi.org/10.1038/nsmb.2133

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