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Related Experiment Videos

T7 RNA polymerase: conformation, functional groups, and promotor binding.

J L Oakley, J A Pascale, J E Coleman

    Biochemistry
    |October 21, 1975
    PubMed
    Summary

    T7 RNA polymerase activity requires a specific thiol group and intact tyrosyl residues for DNA binding and transcription. Enzyme structure remains stable under various conditions, but DNA fragmentation affects RNA synthesis patterns.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Enzymology

    Background:

    • T7 RNA polymerase is a crucial enzyme for gene expression in bacteriophage T7.
    • Understanding its structural and functional properties is essential for elucidating transcription mechanisms.

    Purpose of the Study:

    • To investigate the structural stability of T7 RNA polymerase under various conditions.
    • To identify key functional residues and their roles in DNA binding and catalysis.
    • To analyze the impact of DNA template modification on transcription.

    Main Methods:

    • Circular dichroism spectroscopy to assess secondary structure.
    • Nitrocellulose filter binding assays to measure DNA and nucleotide binding.
    • Chemical modification (alkylation, nitration) to probe functional residues.

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  • Restriction enzyme digestion and gel electrophoresis to analyze transcription products.

    • Main Results:

    • T7 RNA polymerase secondary structure (approx. 12% alpha helix) is stable across a range of conditions.
    • A single thiol (-SH) group is essential for catalytic activity but not DNA binding.
    • Nitration of surface tyrosyl residues abolishes DNA binding.
    • DNA fragmentation by Hpa II alters transcription patterns, producing shorter mRNAs, while promoter regions remain intact.

    Conclusions:

    • T7 RNA polymerase possesses a stable secondary structure.
    • Specific amino acid residues (thiol and tyrosyl) are critical for enzyme function.
    • DNA template integrity influences the pattern of RNA synthesis, highlighting the importance of promoter accessibility.