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Purification and properties of two RNA polymerases from Physarum polycephalum

A B Burgess, R R Burgess

    Proceedings of the National Academy of Sciences of the United States of America
    |April 1, 1974
    PubMed

    Insights

    Researchers purified two RNA polymerases from slime mold, one sensitive and one resistant to alpha-amanitin. The resistant enzyme, with two subunits, functions optimally at low ionic strength, unlike its sensitive counterpart.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Mycology

    Background:

    • Slime molds, such as Physarum polycephalum, are eukaryotic organisms with unique biological processes.
    • Understanding RNA polymerase function is crucial for gene expression regulation in all life forms.

    Purpose of the Study:

    • To purify and characterize RNA polymerases from Physarum polycephalum.
    • To investigate the properties and subunit composition of alpha-amanitin-sensitive and resistant RNA polymerases.

    Main Methods:

    • Purification of RNA polymerases using biochemical techniques.
    • Enzyme activity assays with varying DNA templates and divalent cations (Mn++ vs. Mg++).
    • Ionic strength optimization experiments.
    • Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for subunit analysis.

    Main Results:

    • Two distinct RNA polymerases were isolated: one sensitive and one resistant to alpha-amanitin.
    • Both enzymes exhibited higher activity with denatured DNA compared to native DNA.
    • Manganese ions (Mn++) were preferred over magnesium ions (Mg++) for both enzymes.
    • The alpha-amanitin-sensitive enzyme's optimal activity was at 0.15 M KCl.
    • The alpha-amanitin-resistant enzyme showed peak activity at very low ionic strength.
    • SDS-PAGE revealed the resistant enzyme comprises two subunits (205,000 and 125,000 Da) in a 1:1 ratio.

    Conclusions:

    • Physarum polycephalum possesses distinct RNA polymerases with differential sensitivity to alpha-amanitin.
    • These enzymes display unique substrate preferences and ionic strength optima, suggesting specialized roles in transcription.
    • The subunit composition of the resistant RNA polymerase provides insights into its structure and function.

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