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Real-time assembly landscape of bacterial 30S translation initiation complex

Nature Structural & Molecular Biology volume 19, pages 609–615 (2012)Cite this article

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Abstract

Initiation factors guide the ribosome in the selection of mRNA and translational reading frame. We determined the kinetically favored assembly pathway of the 30S preinitiation complex (30S PIC), an early intermediate in 30S initiation complex formation in Escherichia coli. IF3 and IF2 are the first factors to arrive, forming an unstable 30S–IF2–IF3 complex. Subsequently, IF1 joins and locks the factors in a kinetically stable 30S PIC to which fMet-tRNAfMet is recruited. Binding of mRNA is independent of initiation factors and can take place at any time during 30S PIC assembly, depending on the cellular concentration of the mRNA and the structural determinants at the ribosome-binding site. The kinetic analysis shows both specific and cumulative effects of initiation factors as well as kinetic checkpoints of mRNA selection at the entry into translation.

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Figure 1: Translation initiation in bacteria.
Figure 2: Recruitment of IF1 to the 30S PIC.
Figure 3: Recruitment of IF2 to the 30S PIC.
Figure 4: Recruitment of IF3 to the 30S PIC.
Figure 5: Recruitment of IF3 to ribosome recycling complexes.
Figure 6: mRNA recruitment.
Figure 7: Assembly landscape of 30S PIC formation.

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Acknowledgements

We thank W. Wintermeyer for critically reading and shortening the manuscript, C. Kothe for doing some stopped-flow experiments and O. Geintzer, S. Kappler, C. Kothe and T. Wiles for expert technical assistance. We also thank the participants of the methods course on transient kinetics of the Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences for doing some stopped-flow experiments. This work was supported by grants of the Deutsche Forschungsgemeinschaft (M.V.R.) and Italian Ministero dell′Instruzione, dell′Universitá e della Ricerca (C.O.G.).

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Authors and Affiliations

  1. Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Pohl Milón, Liudmila Filonava & Marina V Rodnina

  2. Department of Biosciences and Biotechnology, Laboratory of Genetics, University of Camerino, Camerino, Italy

    Cristina Maracci & Claudio O Gualerzi

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  1. Pohl Milón
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  2. Cristina Maracci
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  4. Claudio O Gualerzi
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Contributions

P.M., C.O.G. and M.V.R. conceived the research. P.M. and M.V.R. designed the experiments and analyzed the data. P.M., C.M. and L.F. prepared materials and conducted experiments. P.M., C.O.G. and M.V.R. wrote the paper.

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Correspondence to Marina V Rodnina.

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Milón, P., Maracci, C., Filonava, L. et al. Real-time assembly landscape of bacterial 30S translation initiation complex. Nat Struct Mol Biol 19, 609–615 (2012). https://doi.org/10.1038/nsmb.2285

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