SHAPE-seq reveals the typical 5′mRNA structure of Escherichia coli and its effect on gene expression

Abstract:

[Objective] Due to the short half-life of mRNA transcript, their anti-degradation ability and ability to recruit ribosomes to initiate translation have significant effects on the gene expression in prokaryotes. However, the multiple functional regions in the 5′ end, of mRNA can affect the half-life and thus the expression of target genes, including the Shine-Dalgarno (SD) sequence and its upstream and downstream translational standby sites (TSSs) and the N-terminal coding sequence (NCS). The own and cross-regional structural differences will affect gene expression. Therefore, it is important to analyze the structure-activity relationship of each functional region. [Methods] We employed primer extension sequencing (SHAPE-seq) to analyze the structural characteristics of seven different 5′mRNAs in Escherichia coli and collected the mRNA abundance and protein level information under their regulation. [Results] Under the regulation of unstructured NCS, the mRNA abundance and protein level were increased by 10 and 19 times, respectively. The formation of secondary structure TSS with a stem length of 10 nt increased the mRNA abundance. When the SD sequence was wrapped to form a secondary structure, the efficiency of translation initiation mediated by the SD sequence was affected (protein level downregulation by 10%). The combination of TSS and NCS significantly increased the mRNA abundance and protein level by 11 and 60 folds, respectively. [Conclusion] We characterized the structure of each region of 5′mRNA conducive to prokaryotic gene expression and revealed the structure-activity relationship of each functional region, providing a new regulatory element for target gene expression in industrial microorganisms.

Key words:translational standby site;N-terminal coding sequence;SD sequence;SHAPE-seq;structure-activity relationship