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An Escherichia coli mutant with a temperature-sensitive function affecting bacteriophage Qbeta RNA replication.

N C Mandal, P M Silverman

    Proceedings of the National Academy of Sciences of the United States of America
    |April 1, 1977
    PubMed
    Summary

    A temperature-sensitive Escherichia coli mutant supports bacteriophage Qbeta replication at 33°C but not 40°C. This indicates a specific component is essential for viral RNA replication, not general RNA synthesis.

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    Area of Science:

    • Molecular Biology
    • Virology
    • Microbiology

    Background:

    • Bacteriophage Qbeta replication relies on host factors within Escherichia coli.
    • Understanding temperature-sensitive mutants aids in dissecting essential viral and host processes.

    Purpose of the Study:

    • To isolate and characterize an E. coli mutant with temperature-dependent bacteriophage Qbeta replication.
    • To identify the specific stage of viral RNA replication affected by the temperature-sensitive defect.

    Main Methods:

    • Isolation of temperature-sensitive E. coli mutants.
    • Bacteriophage plaque assays at different temperatures.
    • Temperature-shift experiments post-infection.
    • Analysis of viral RNA synthesis using zone sedimentation.

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    Main Results:

    • A mutant was identified that supports Qbeta replication at 33°C but not 40°C.
    • Qbeta replication initiation was blocked within 20-30 minutes of infection at 40°C.
    • Mutant cells produced partially double-stranded 9S RNA instead of viral 32S RNA at the restrictive temperature.
    • The temperature-sensitive defect did not impede Qbeta replicase synthesis or activity.

    Conclusions:

    • A specific temperature-sensitive host factor is crucial for bacteriophage Qbeta RNA replication, not for replicase function.
    • This factor is essential for the synthesis of full-length viral RNA, distinguishing it from other replicase-catalyzed RNA synthesis.