Influence of 5′-UTR nucleotide composition on translation efficiency in Escherichia coli

Translation initiation for 5'-UTR contributes primarily to the efficient protein expression in Escherichia coli. Many studies have focused on constructing random 5'-UTR libraries to investigate the impact of mRNA features on protein translation efficiency. However, the study on the effect of the absence of specific types of nucleotides in the entire 5'-UTR region on translation efficiency has not yet been reported. Here, we constructed four reporter plasmid libraries encoding the sfGFP fluorescent protein, each preceded by 5'-UTRs that lack one specific nucleotide (25B, 25D, 25H, 25V). Each library was transformed into E. coli cells, and the fluorescence distribution among the different libraries was analyzed by flow cytometer. Additionally, we quantified the activity of 256 unique 5'-UTR sequences and analyzed the impact of the corresponding mRNA sequence features on translation efficiency. We found that the 25D library, which lacks the C nucleotide, exhibited the highest overall translation efficiency compared to the other three libraries. Moreover, the minimum free energy and 16S rRNA hybridization energy of the 5'-UTR sequence could work coordinately to influence translation efficiency. The 5'-UTR sequences lacking the C nucleotide also achieve efficient protein translation. These findings may provide several guiding principles for precisely tuning protein expression.