Post-transcriptional regulation of the str operon in Escherichia coli

In the Escherichia coli str operon, translation of the S12 and S7 genes is largely coupled, and the translational repressor S7 inhibits S7 translation, which is coupled to that of S12, but does not inhibit independent translation of S7 by free ribosomes in the intracellular pool. We have studied the S12-S7 intercistronic region of mRNA by analyzing RNA synthesized in vitro using structure-specific nucleases and a chemical probe, dimethyl sulfate. Based on the results obtained, we have deduced a secondary structure model of the S12-S7 intercistronic region and identified nucleotide residues “protected” by S7. We then carried out site-directed mutagenesis to identify nucleotide residues important for S7 translation as well as for repression by S7. The results showed that two distinct regions are important for S7-mediated repression; one is the S7 binding region identified by the protection analysis and the second is the stem structure that sequesters the Shine-Dalgarno sequence for the S7 gene. Some of the base alterations in the first region abolished S7 binding and, as a consequence, abolished S7-mediated repression, without affecting the efficiency of S7 translation. Other mutations disrupting the stem structure in the second region abolished S7-mediated repression without significantly affecting the S7-mRNA interaction. We also found that certain mutations drastically decrease S7 translation achieved by translational coupling without affecting S7 translation achieved by independent initiation. These mutations are in base-paired regions and evidence was obtained to suggest that these base-paired structures are important for translational coupling. We suggest that some specific RNA structures in the intercistronic region play an active role in achieving translational coupling in this system, and that repression of S7 translation by S7 protein is due to disruption of such structures induced by binding of S7 protein to the target site, rendering translational coupling very inefficient, but leaving independent translation initiation unaffected.