Translation Enhancement by a Short Nucleotide Insertion at 5′UTR: Application to an In Vitro Cell-Free System and a Photosynthetic Bacterium

Abstract

We previously showed that insertion of Dictyostelium gene sequences, such as mlcR, upstream of the Shine–Dalgarno sequence, positively impacts downstream gene expression in Escherichia coli. However, the mechanism by which protein production is facilitated and its applicability to other bacteria remains unknown. In this study, a translation-enhancing effect, associated with this system, on the mRNA amount and property as well as the versatility of the method has been demonstrated. The insertion of mlcR-terminal 25 bp (mlcR25) stabilized the mRNAs and led to increased mRNA levels in E. coli. In the in vitro translation system, a four-fold enhancement was observed when DNA was used as the template, and a three-fold enhancement was observed when mRNA was used as the template. This suggests that mlcR25 has an effect on the facilitation of the interaction between mRNA and ribosome. Furthermore, when this enhancement system was adapted to the photosynthetic bacterium Rhodobacter capsulatus, a more than six-fold increase in translation was observed. Thus, we propose that enhanced translation by mlcR25 is mediated by mechanisms that help the translation machinery to work efficiently, and the system can be applied to bacteria other than E. coli.