Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome

Abstract

Initiation of protein synthesis is a universally conserved event that requires initiation factors IF1, IF2 and IF3 in prokaryotes. IF2 is a GTPase essential for binding initiator transfer RNA to the 30S ribosomal subunit and recruiting the 50S subunit into the 70S initiation complex. We present two cryo-EM structures of the assembled 70S initiation complex comprising mRNA, fMet-tRNAfMet and IF2 with either a non-hydrolyzable GTP analog or GDP. Transition from the GTP-bound to the GDP-bound state involves substantial conformational changes of IF2 and of the entire ribosome. In the GTP analog–bound state, IF2 interacts mostly with the 30S subunit and extends to the initiator tRNA in the peptidyl (P) site, whereas in the GDP-bound state IF2 steps back and adopts a 'ready-to-leave' conformation. Our data also provide insights into the molecular mechanism guiding release of IF1 and IF3.

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Figure 1: Structure of the T. thermophilus mRNA–fMet-tRNAfMet–IF2–GMPPCP–70S ribosome complex showing the binding site and the interaction pattern of IF2.
Figure 2: Structure of the T. thermophilus mRNA–fMet-tRNAfMet–IF2–GDP–70S ribosome complex showing the binding site and the interaction pattern of IF2 as in Figure 1.
Figure 3: Comparison of the GTP- and GDP-bound states of IF2, illustrating the conformational change and spatial shift of IF2 and the associated conformational change of the ribosome upon GTP hydrolysis.

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Acknowledgements

We thank J. Thompson (Brown University, Providence, Rhode Island, USA) for providing IF2 strains and D. Moras, P. Schultz, J.-C. Thierry and V. Mallouh for sustained support. We are grateful to L. Jenner for comments on the manuscript and to M. Schatz and R. Schmidt for support with the IMAGIC-5 software. A.G.M. and S.M. are recipients of a postdoctoral fellowship from the CNRS, and A.S. benefited from a fellowship from the Camerino University exchange program. The electron microscope was financed by the Alsace Region, the INSERM, the CNRS and the Association pour la Recherche sur le Cancer.

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Correspondence to Bruno P Klaholz.

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Supplementary information

Supplementary Fig. 1

Pair-wise sequence alignment between T. Thermophilus and M thermoautotrophicum IF2/eIF5B sequences. (PDF 228 kb)

Supplementary Fig. 2

Stereo representations of hypothetical models concerning release of IF1 and IF2 during translation initiation. (PDF 791 kb)

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Myasnikov, A., Marzi, S., Simonetti, A. et al. Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome. Nat Struct Mol Biol 12, 1145–1149 (2005). https://doi.org/10.1038/nsmb1012

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