Evaluation of synthetic post-transcription regulatory sequences reveals design principle to enhance mRNA stability and translation efficiency

The Shine-Dalgarno (SD) sequence and its adjacent flanking regions, including the translation standby site (TSS) and the N-terminal coding sequence (NCS), play critical roles in regulating ribosome recruitment, translation efficiency, and mRNA stability against RNase degradation. However, structure-activity relationships governing these regions remain poorly characterized, and their functional interplay introduces substantial complexity. In this study, we employed one-pot technology to build a post-transcriptional regulatory component (PTRC) library of 576 variants to clarify the relationship between sequence variants and both protein expression and mRNA levels via high-throughput sequencing. Our results show that although unstructured TSSs do not enhance mRNA stability, they markedly increase translation efficiency, causing a 16 %-100 % rise in protein expression. In contrast, structured TSSs increase mRNA levels by 43 %-90 %. Additionally, highly conserved SD sequences boost translation efficiency by up to 10 % and mRNA abundance by up to 12 %. Moreover, it was found that optimized linear N-terminal coding sequence (NCS) positively affects protein expression and mRNA levels. The effects of these optimized regulatory components were verified in the expression control of the <i>nrk</i> and <i>sam2</i> genes, resulting in enhanced production. These findings underscore the crucial role of structural optimization, guiding the rational design of synthetic post-transcriptional regulatory elements.