Influence of 5’-UTR base types on translation efficiency in Escherichia coli

Abstract

Objectives Translation initiation for 5’ untranslated regions (5’-UTR) contributes primarily to the efficient expression of proteins in Escherichia coli (E. coli). Many studies have focused on the construction of random 5’-UTR libraries to explore the effects of mRNA features on protein translation efficiency. However, the study on the effect of the lack of specific types of bases in the entire 5’-UTR region on translation efficiency has not been reported yet. Results We constructed four reporter plasmid libraries encoding sfGFP fluorescent protein preceded by 5’-UTRs lacking one type of base (25B, 25D, 25H, 25V). Each library was transformed into E. coli BL21 Star (DE3) cells and the fluorescence distribution among the different libraries were analyzed by flow cytometer (FCM). In addition, we quantified the activity of 256 unique 5’-UTR sequences and analyzed the effect of the corresponding mRNA sequence features on translation efficiency. We found that the 25D library, which lacks the C base, had the highest overall translation efficiency compared to the other three libraries. Moreover, minimum free energy (MFE) and 16S rRNA hybridization energy of the 5’-UTR sequence could work coordinately to influence translation efficiency. Conclusions The 5’-UTR sequences lacking the C base also achieve efficient protein translation. These findings could provide several guiding principles for the precise tuning of protein expression.