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Ribosomal Protein S5e is Implicated in Translation Initiation through its Interaction with the N-Terminal Domain of Initiation Factor eIF2α

Authors

  • Dmitri Sharifulin,

    1. Laboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk, 630090 (Russia)
    2. Department of Molecular Biology, Novosibirsk State University, Ulitsa Pirogova 2, Novosibirsk, 630090 (Russia)
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  • Dr. Elena Babaylova,

    1. Laboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk, 630090 (Russia)
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  • Dr. Olga Kossinova,

    1. Laboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk, 630090 (Russia)
    2. Department of Molecular Biology, Novosibirsk State University, Ulitsa Pirogova 2, Novosibirsk, 630090 (Russia)
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  • Dr. Yulia Bartuli,

    1. Laboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk, 630090 (Russia)
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  • Dr. Dmitri Graifer,

    1. Laboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk, 630090 (Russia)
    2. Department of Molecular Biology, Novosibirsk State University, Ulitsa Pirogova 2, Novosibirsk, 630090 (Russia)
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  • Prof. Galina Karpova

    Corresponding author
    1. Laboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk, 630090 (Russia)
    2. Department of Molecular Biology, Novosibirsk State University, Ulitsa Pirogova 2, Novosibirsk, 630090 (Russia)
    • Laboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Prospekt Lavrentieva 8, Novosibirsk, 630090 (Russia)

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Abstract

A key step of translation initiation in eukaryotes is formation of the 48S preinitiation complex (PIC) containing the 40S ribosome, a set of eukaryotic initiation factors (eIFs), mRNA, and initiator Met-tRNA interacting with mRNA start codon; however, the PIC structure remains substantially unknown. Here, we apply formaldehyde-induced protein–protein crosslinks to identify contacts between ribosomal protein S5e (rpS5e, “e” stands for “eukaryotic”) and eIFs within the mammalian PIC, assembled on either model canonical or IRES-containing mRNA. Using immunoblotting and mass spectrometry, we show that with both types of mRNA, rpS5e crosslinks to eIF2α. Comparative analysis of peptides resulting from trypsinolysis of the crosslinked proteins before and after crosslink reversal reveals crosslinked peptides in the N-terminal parts of rpS5e and eIF2α. Application of these data to a model PIC structure obtained with the use of available structures indicates that eIF2α undergoes major conformation rearrangements to enable contacts of the factor with rpS5e. These contacts are suggested to maintain the correct positioning of eIF2α relative to other PIC components; this could be essential for start-codon selection by the PIC.

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