Translation Enhancement by a Short Nucleotide Insertion at 5′UTR: Application to an In Vitro Cell-Free System and a Photosynthetic Bacterium
2023
Tomo Kondo | Takayuki Shimizu
We previously showed that insertion of <i>Dictyostelium</i> gene sequences, such as <i>mlcR</i>, upstream of the Shine–Dalgarno sequence, positively impacts downstream gene expression in <i>Escherichia coli</i>. 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 <i>mlcR</i>-terminal 25 bp (<i>mlcR25</i>) stabilized the mRNAs and led to increased mRNA levels in <i>E. coli</i>. 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 <i>mlcR25</i> has an effect on the facilitation of the interaction between mRNA and ribosome. Furthermore, when this enhancement system was adapted to the photosynthetic bacterium <i>Rhodobacter capsulatus</i>, a more than six-fold increase in translation was observed. Thus, we propose that enhanced translation by <i>mlcR25</i> is mediated by mechanisms that help the translation machinery to work efficiently, and the system can be applied to bacteria other than <i>E. coli</i>.
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