Biomedical subjects
N F Habib
Publications and source records attributed to N F Habib.
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.
Identification of a B subunit gene promoter in the Shiga toxin operon of Shigella dysenteriae 1.
The Shiga toxin operon (stx) is composed of A and B subunit genes which are transcribed as a bicistronic mRNA from a promoter which lies 5' to the stxA gene. Northern (RNA) blot and primer extension analyses revealed the existence of a second stxB gene transcript. Recombinant plasmids which carried the stxB gene without the stx operon promoter and with the influence of a vector promoter abrogated produced STX B polypeptides, suggesting that the stxB gene mRNA was transcribed from an independent promoter and was not produced by endoribonucleotic processing of the bicistronic mRNA. Examination of the DNA sequences 5' to the stxB gene transcription initiation site which were carried by the recombinant plasmids revealed a region with high homology to the consensus for Escherichia coli promoters. Deletion and mutation of this region affected StxB and holotoxin production, establishing its role in the regulation of the stxB gene. Comparison of the promoters by using a transcription analysis vector revealed that the stxB gene promoter differed from the stx operon promoter in that was approximately sixfold less efficient and was not repressed by iron. Identification of a second promoter in the stx operon indicates that independent transcription of the stxB gene may regulate overproduction of the STX B polypeptides and may contribute to the 1A:5B subunit stoichiometry of the holotoxin.