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Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs
10:37

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Published on: May 10, 2018

ATP-dependent unwinding of messenger RNA structure by eukaryotic initiation factors

B K Ray, T G Lawson, J C Kramer

    The Journal of Biological Chemistry
    |June 25, 1985
    PubMed

    Insights

    Eukaryotic initiation factors (eIFs) unwind mRNA structure for translation. Eukaryotic initiation factor 4F (eIF-4F) is a more efficient unwinding factor than eIF-4A, with its activity localized to a specific polypeptide.

    Area of Science:

    • Molecular Biology
    • Protein Synthesis
    • Gene Expression Regulation

    Background:

    • Early events in eukaryotic translation involve the interaction of protein synthesis initiation factors with messenger RNA (mRNA).
    • Understanding these interactions is crucial for characterizing the eukaryotic translation pathway.
    • Reovirus mRNAs and specific eukaryotic initiation factors (eIFs) were utilized to investigate these early events.

    Purpose of the Study:

    • To characterize the early events in eukaryotic translation by studying the interaction of protein synthesis initiation factors with mRNA.
    • To determine the roles of eIF-4A, eIF-4B, and eIF-4F in mRNA structure modulation.
    • To elucidate the mechanism and efficiency of mRNA unwinding by these factors.

    Main Methods:

    • Utilized reovirus mRNAs labeled with 32P.
    • Employed purified eukaryotic initiation factors (eIF)-4A, -4B, and -4F from rabbit reticulocytes.
    • Assessed mRNA structural changes using nuclease sensitivity tests.
    • Investigated the requirement of ATP and the effect of m7GDP.

    Main Results:

    • Eukaryotic initiation factor 4A (eIF-4A) induces mRNA structural changes, suggesting an mRNA unwinding capability dependent on ATP.
    • Eukaryotic initiation factor 4B (eIF-4B) enhances the unwinding activity of eIF-4A at lower concentrations.
    • Eukaryotic initiation factor 4F (eIF-4F) exhibits similar mRNA unwinding activity, being over 20-fold more efficient than eIF-4A and inhibited by m7GDP, indicating its activity is cap-dependent and localized to a specific polypeptide.

    Conclusions:

    • Eukaryotic initiation factors play a critical role in modulating mRNA structure for translation initiation.
    • eIF-4A unwinds mRNA structure, an activity enhanced by eIF-4B.
    • eIF-4F is a highly efficient mRNA unwinding complex, with its activity likely residing in the 46,000-dalton polypeptide, and is involved in cap-dependent translation initiation.

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