Vitamin B12 repression of the cob operon in Salmonella typhimurium: translational control of the cbiA gene

Abstract

Expression of the cob operon is repressed by B12 via a post-transcriptional control mechanism which requires sequence elements within the leader region of the mRNA and the first gene of the operon, the cbiA gene. Here we show that B12 repression of cbiA gene expression occurs at the level of translation initiation through sequestration of the ribosomal binding site (rbs) in an RNA hairpin. Analysis of mutations that destabilize or restabilize the secondary structure demonstrates that folding of the hairpin is essential for repression. The existence of the hairpin was confirmed by a secondary structure analysis of RNA from the wild type and three mutants. Deletions that remove the upstream part of the leader confer a drastic reduction in translation efficiency. This low-level translation is caused by the hairpin, as indicated by the finding that suppressor mutations that destabilize the hairpin restore efficient translation. Thus, the native upstream RNA functions as a translation enhancer and acts to relieve the hairpin's inhibitory effect on translation initiation. The inhibitory effect of the hairpin was confirmed by a ribosomal toeprinting analysis. We propose that the translational control of the cbiA gene mediates repression of the entire cob operon.