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Related Experiment Videos

Eukaryotic mRNA capping enzyme-guanylate covalent intermediate.

S Venkatesan, B Moss

    Proceedings of the National Academy of Sciences of the United States of America
    |January 1, 1982
    PubMed
    Summary
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    HeLa cell mRNA guanylyltransferase forms a covalent enzyme-guanylate complex with GTP. This complex can transfer GMP to form GTP or a cap structure on poly(A) RNA.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Enzymology

    Background:

    • Messenger RNA (mRNA) capping is crucial for mRNA stability and translation initiation in eukaryotes.
    • mRNA guanylyltransferase (EC 2.7.7.50) is the enzyme responsible for adding the guanine nucleotide cap to the 5' end of nascent mRNA.
    • Understanding the mechanism of mRNA guanylyltransferase is vital for comprehending gene expression regulation.

    Purpose of the Study:

    • To investigate the formation and properties of the covalent enzyme-guanylate complex of HeLa cell mRNA guanylyltransferase.
    • To elucidate the nature of the linkage between GMP and the enzyme.
    • To characterize the transfer capabilities of the enzyme-guanylate complex.

    Main Methods:

    • Incubation of HeLa cell mRNA guanylyltransferase with [alpha-32P]GTP and a divalent cation.

    Related Experiment Videos

  • Purification of the covalent complex using phosphocellulose chromatography.
  • Analysis of the GMP-polypeptide linkage stability (alkali, acid, hydroxylamine).
  • Enzymatic assays for GMP transfer to pyrophosphate and poly(A).
  • Peptide mapping after trypsin digestion and 2D electrophoresis.

    • Main Results:

    • A covalent enzyme-guanylate complex was formed upon incubation of mRNA guanylyltransferase with [alpha-32P]GTP.
    • The purified complex transferred GMP to pyrophosphate (regenerating GTP) or to the 5'-diphosphate end of poly(A) to form a cap structure.
    • The GMP-polypeptide complex exhibited a molecular weight of 65,000.
    • The linkage was identified as a phosphoamine bond, characterized by alkali stability, acid lability, and susceptibility to nucleophilic attack.
    • Trypsin digestion yielded a single GMP-peptide resolved by 2D electrophoresis.

    Conclusions:

    • HeLa cell mRNA guanylyltransferase forms a stable covalent intermediate with GMP during the capping process.
    • The enzyme-guanylate linkage is a phosphoamine bond, crucial for the transfer of GMP to RNA.
    • This study provides mechanistic insights into mRNA cap formation by guanylyltransferase.