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Journal of Bacteriology, March 1999, p. 1811-1819, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Analysis of Elements Involved in Pseudoknot-Dependent Expression and Regulation of the repA Gene of an IncL/M Plasmid

V. Athanasopoulos, J. Praszkier, and A. J. Pittard*

Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3052, Australia

Received 13 August 1998/Accepted 28 December 1998

Replication of the IncL/M plasmid pMU604 is controlled by a small antisense RNA molecule (RNAI), which, by inhibiting the formation of an RNA pseudoknot, regulates translation of the replication initiator protein, RepA. Efficient translation of the repA mRNA was shown to require the translation and correct termination of the leader peptide, RepB, and the formation of the pseudoknot. Although the pseudoknot was essential for the expression of repA, its presence was shown to interfere with the translation of repB. The requirement for pseudoknot formation could in large part be obviated by improving the ribosome binding region of repA, either by replacing the GUG start codon by AUG or by increasing the spacing between the start codon and the Shine-Dalgarno sequence (SD). The spacing between the distal pseudoknot sequence and the repA SD was shown to be suboptimal for maximal expression of repA.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3052, Australia. Phone: 61 3 9344 7751. Fax: 61 3 9347 1540. E-mail: aj.pittard{at}microbiology.unimelb.edu.au.

Journal of Bacteriology, March 1999, p. 1811-1819, Vol. 181, No. 6
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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