Replication control of plasmid R1: disruption of an inhibitory RNA structure that sequesters the repA ribosome-binding site permits tap-independent RepA synthesis

Authors

  • Pontus Blomberg,

    1. Department of Microbiology, Biomedical Center, Uppsala University, Box 581, S-75123 Uppsala, Sweden.
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    • Division of Basic Sciences, 1124 Columbia Street, A2-168, Fred Hutchinson Cancer Research Center, Seattle, Washington 98119, USA.

    • These authors contributed equally to the work presented.

  • Hilde M. Engdahl,

    1. Department of Microbiology, Biomedical Center, Uppsala University, Box 581, S-75123 Uppsala, Sweden.
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    • These authors contributed equally to the work presented.

  • Charlotta Malmgren,

    1. Department of Microbiology, Biomedical Center, Uppsala University, Box 581, S-75123 Uppsala, Sweden.
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    • These authors contributed equally to the work presented.

  • Pascale Romby,

    1. UPR du CNRS no. 9002, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, 67084 Strasbourg, France.
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  • E. Gerhart H. Wagner

    Corresponding author
    1. Department of Microbiology, Biomedical Center, Uppsala University, Box 581, S-75123 Uppsala, Sweden.
    • *For correspondence. Tel. (18) 174527; Fax (18) 530396.

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Summary

The replication frequency of plasmid R1 is controlled by an antisense RNA, CopA, that inhibits the synthesis of the replication initiator protein, RepA, at the post-transcriptional level. This inhibition is indirect and affects translation of a leader peptide reading frame (tap). Translation of tap is required for repA translation (Blomberg et al., 1992). Here we asked whether an RNA stem-loop sequestering the repA ribosome-binding site blocks tap translation-independent repA expression. Destabilization of this structure resulted in tap-independent RepA synthesis, concomitant with a loss of CopA-mediated inhibition; thus, CopA acts at the level of tap translation. Structure probing of RepA mRNAs confirmed that the introduced mutations induced a local destabilization in the repA ribosome-binding site stem-loop. An increased spacing between the repA Shine-Dalgarno region and the start codon permitted even higher rep A expression. In Incα/IncB plasmids, an RNA pseudoknot acts as an activator for rep translation. We suggest that the regulatory pathway in plasmid R1 does not involve an activator RNA pseudoknot.

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