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Roles of a ribosome-binding site and mRNA secondary structure in differential expression of Shiga toxin genes.

Department of Immunology and Microbiology, Wayne State University School of Medicine, Detroit, Michigan 48201.

ABSTRACT

The Shiga toxin operon (stx) is composed of two genes for the A and B subunits, which are transcribed from a promoter 5' to the stxA gene. The 1A:5B subunit stoichiometry of the holotoxin suggests that the stxA and stxB genes are differentially regulated. In a previous study, we demonstrated the existence of a second promoter which independently transcribes the stxB gene. However, transcription fusion analysis revealed that the independent stxB gene promoter is not solely responsible for a fivefold increase in B polypeptide production. In this study, we have investigated the role of an independent stxB gene ribosome-binding site (RBS) in the overexpression of STX B subunits. Site-directed mutagenesis was used to eliminate this RBS and establish its role in StxB production. Examination of the nucleotide sequences surrounding the stxB gene RBS revealed a potential for the formation of a stem-loop structure with a calculated delta G of -7.563 kcal/mol (ca. -31.64 kJ/mol). Sequences surrounding the stxA gene RBS were found not to possess a similar potential for secondary-structure formation. Disruption of the stem-loop surrounding the stxB gene RBS by 2- and 4-nucleotide substitutions caused a significant reduction in B polypeptide and holotoxin production, establishing the role of this secondary structure in the enhancement of translation of the stxB gene.

This article has been cited by other articles:

• Paton, J. C., Paton, A. W. (1998). Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections. Clin. Microbiol. Rev. 11: 450-479 [Abstract] [Full Text]