The secondary structure of the 5’-end of the STb mRNA affects its secretion efficiency

The majority of secreted proteins in E. coli are targeted to and translocated across the cytoplasmic membrane through a signal sequence within the N-terminus of the protein. The major membrane complex facilitating this process is the general secretory or Sec-dependent membrane complex, also known as T2SS secretion. Research with proteins that are processed for export through a different pathway, called the T3SS secretion system, has recently challenged this widely accepted hypothesis. This research has provided evidence that the T3SS secretion system substrates may use mRNA sequences as a secondary means of protein targeting in addition to the N-terminus of the synthesized protein. The main objective of this study was to test whether or not the mRNA of the T2SS secretion system substrates may have structural information sufficient for efficient targeting of the protein for secretion. A well-studied substrate of the T2SS secretion system in E. coli is the heat-stable enterotoxin B (STb). The STb gene was cloned and a number of mutations were introduced within the 5' end of its mRNA to look at the effect of such mutations on protein secretion. The results showed that mutations that lead to changes in the AT/GC ratio within this 5’ region of the gene, while conserving the amino acid sequence of that region, affected toxin secretion. Bioinformatic analysis showed that these mutations affected the secondary structure of the 5' end of the mRNA and indicated that significant changes can be produced in the secondary structure of the mRNA when the AT/GC ratio is modified. This data shows that alterations in the secondary structure of the 5' end of the mRNA, without changes in the amino acid sequence of the N-terminus of the protein, can affect the efficiency with which the toxin is secreted and hints to a possible role for the mRNA in targeting proteins for export across the cytoplasmic membrane.