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Conformational selection of translation initiation factor 3 signals proper substrate selection

Nature Structural & Molecular Biology volume 20, pages 628–633 (2013)Cite this article

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Abstract

During translation, initiation factor 3 (IF3) binds to the small (30S) ribosomal subunit and regulates the fidelity with which the initiator tRNA and mRNA start codon substrates are selected into the 30S initiation complex (30S IC). The molecular mechanism through which IF3 promotes the recognition and signaling of correct substrate selection, however, remains poorly defined. Using single-molecule fluorescence resonance energy transfer, we show that 30S IC–bound Escherichia coli IF3 exists in a dynamic equilibrium between at least three conformations. We found that recognition of a proper anticodon-codon interaction between initiator tRNA and the start codon within a completely assembled 30S IC selectively shifts this equilibrium toward a single conformation of IF3. Our results strongly support a conformational selection model in which the conformation of IF3 that is selectively stabilized within a completely and correctly assembled 30S IC facilitates further progress along the initiation pathway.

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Figure 1: Translation initiation in bacteria and fluorescent labeling of IF3.
Figure 2: smFRET measurements of , , and 30S IC−tRNA.
Figure 3: smFRET measurements of 30S IC−tRNA, 30S ICfMet, 30S ICPhe, 30S ICLys, 30S ICPhe,UUC and 30S ICfMet,AUU.
Figure 4: Observation of an intermolecular IF3–50S subunit smFRET signal.
Figure 5: A structure-based mechanistic model for how IF3 recognizes and signals fMet-tRNAfMet and start-codon selection within the 30S IC.

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Acknowledgements

We thank M.A. Gawinowicz for performing trypsin digestion and MALDI-TOF mass spectrometry analysis of IF3(Cy3-Cy5), D. MacDougall for assistance with protein purification and J.W. van de Meent for assistance with smFRET data analysis. We also thank members of the Gonzalez research group, especially D. MacDougall, J. Wang, K. Caban and C. Kinz-Thompson, for discussions and comments on the manuscript. Financial support for this work was provided by Career Award in the Biomedical Sciences 1004856 from the Burroughs Wellcome Fund (R.L.G.), Research Project Grant GM084288 from the US National Institutes of Health (R.L.G.) and Molecular Biophysics Training Grant T32GM008281 from the US National Institutes of Health (M.M.E.).

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  1. Margaret M Elvekrog

    Present address: Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA.,

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  1. Department of Chemistry, Columbia University, New York, New York, USA

    Margaret M Elvekrog & Ruben L Gonzalez Jr

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M.M.E. and R.L.G. contributed to the experimental design, data interpretation and manuscript writing. M.M.E. performed the experiments and carried out data analyses.

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Correspondence to Ruben L Gonzalez Jr.

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The authors declare no competing financial interests.

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Supplementary Figures 1–5, Supplementary Tables 1–5 and Supplementary Note (PDF 5372 kb)

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Elvekrog, M., Gonzalez, R. Conformational selection of translation initiation factor 3 signals proper substrate selection. Nat Struct Mol Biol 20, 628–633 (2013). https://doi.org/10.1038/nsmb.2554

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