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Translational interference at overlapping reading frames in prokaryotic messenger RNA.

B Berkhout, R A Kastelein, J van Duin

    Gene
    |January 1, 1985
    PubMed
    Summary

    Translation of upstream genes can block downstream gene initiation in prokaryotes. This ribosome-binding site (RBS) interference is overcome by mutations in the upstream gene, allowing downstream translation.

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Microbiology

    Background:

    • Ribosome-binding sites (RBS) are crucial for initiating protein synthesis in prokaryotes.
    • In overlapping reading frames, the downstream RBS can be embedded within the upstream coding sequence.
    • The functional impact of this overlap on translation initiation remains an area of investigation.

    Purpose of the Study:

    • To investigate if rapid translation of an upstream gene can prevent initiation at an overlapping ribosome-binding site (RBS).
    • To determine if an actively translated coding sequence can sterically hinder downstream translation initiation.
    • To explore the regulatory mechanisms governing gene expression in overlapping reading frames.

    Main Methods:

    • Utilized the RNA phage MS2 system, known for its overlapping gene structures, as a model.

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  • Employed nonsense mutations in the upstream gene to disrupt its translation.
  • Assessed the translation of the downstream gene under conditions of intact and disrupted upstream gene expression.

    • Main Results:

    • Translation of the upstream cistron effectively blocked initiation of protein synthesis at the overlapping downstream RBS.
    • Restoration of downstream gene translation was observed when nonsense mutations were introduced into the upstream gene.
    • This demonstrates a mechanism of translational coupling and interference in prokaryotic gene expression.

    Conclusions:

    • The high rate of translation of an upstream gene can physically preclude the utilization of an overlapping RBS.
    • This finding provides insight into how prokaryotes regulate gene expression from polycistronic mRNAs with overlapping reading frames.
    • Mutational inactivation of the upstream gene releases the block on downstream translation, highlighting a regulatory switch.