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

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.