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Complementary charge-based interaction between the ribosomal-stalk protein L7/12 and IF2 is the key to rapid subunit association
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.ORCID iD: 0000-0003-2091-0610
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2018 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 18, p. 4649-4654Article in journal (Refereed) Published
Abstract [en]

The interaction between the ribosomal-stalk protein L7/12 (L12) and initiation factor 2 (IF2) is essential for rapid subunit association, but the underlying mechanism is unknown. Here, we have characterized the L12–IF2 interaction on Escherichia coli ribosomes using site-directed mutagenesis, fast kinetics, and molecular dynamics (MD) simulations. Fifteen individual point mutations were introduced into the C-terminal domain of L12 (L12-CTD) at helices 4 and 5, which constitute the common interaction site for translational GTPases. In parallel, 15 point mutations were also introduced into IF2 between the G4 and G5 motifs, which we hypothesized as the potential L12 interaction sites. The L12 and IF2 mutants were tested in ribosomal subunit association assay in a stopped-flow instrument. Those amino acids that caused defective subunit association upon substitution were identified as the molecular determinants of L12–IF2 interaction. Further, MD simulations of IF2 docked onto the L12-CTD pinpointed the exact interacting partners—all of which were positively charged on L12 and negatively charged on IF2, connected by salt bridges. Lastly, we tested two pairs of charge-reversed mutants of L12 and IF2, which significantly restored the yield and the rate of formation of the 70S initiation complex. We conclude that complementary charge-based interaction between L12-CTD and IF2 is the key for fast subunit association. Considering the homology of the G domain, similar mechanisms may apply for L12 interactions with other translational GTPases.

Place, publisher, year, edition, pages
2018. Vol. 115, no 18, p. 4649-4654
Keywords [en]
protein synthesis, ribosomal protein L7/12, protein-protein interaction, ribosome, translation initiation
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:uu:diva-350185DOI: 10.1073/pnas.1802001115ISI: 000431119600050PubMedID: 29686090OAI: oai:DiVA.org:uu-350185DiVA, id: diva2:1204315
Funder
Swedish Research Council, 2014-4423; 2016-06264Knut and Alice Wallenberg Foundation, 2011.0081VINNOVA, 2013-8778Available from: 2018-05-07 Created: 2018-05-07 Last updated: 2018-07-13Bibliographically approved

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Ge, XueliangMandava, Chandra SekharLind, ChristofferÅqvist, JohanSanyal, Suparna

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