Upstream ribosome impediments activate roles of internal Shine-Dalgarno sequence for translation initiation in E. coli

The initiation of protein translation, one of the four phases of translation, had been proved to be the rate limiting step for translation. The specific interaction of bacterial 30S ribosomal subunit with Shine-Dalgarno sequence (SD) contributes the initiation significantly. It had been shown that SD-like sequence in coding region of mRNA, designated as internal SD, can direct translation initiation as regular SD does. The ribosome impediments were demonstrated to be one of the factors contributing the non-uniform translation elongation rate, but their effects on internal SD translation initiation role (ISTIR) remains unclear. To investigate effects of upstream ribosome impediments on ISTIR, a fragment consisting of pyruvate kinase (PK) gene, translation initiation needed mRNA elements (including an A/U-rich region, SD sequence and start codon AUG) and red fluorescent protein (RFP) gene was constructed and RFP expression levels representing translation initiation efficiency induced by internal SD was analyzed. Surprisingly, RFP expression was not detected with this primary construct, further experiments of inclusion of stem loop structure preceding to internal SD or co-expression of engineered RNA binding scaffold (ERBS) targeting to preceding sequence of internal SD could activate ISTIR. These results suggest that upstream ribosome impediments would activate ISTIR to initiate the downstream gene translation, which manifests the potential for developing new method to test interactions between RNA binding proteins and their target RNA molecules in vivo.