mRNA degradation is a central process that affects all gene expression levels, and yet the determinants that control mRNA decay rates remain poorly characterized. Here, we applied a synthetic biology, learn-by-design approach to elucidate the sequence and structural determinants that control mRNA stability in bacterial operons. We designed, constructed, and characterized 82 operons, systematically varying RNAse binding site characteristics, translation initiation rates, and transcriptional terminator efficiencies in the 5' UTR, intergenic, and 3' UTR regions, and measuring their mRNA levels using RT-qPCR assays. We show that introducing long single-stranded RNA into 5' UTRs reduced mRNA levels by up to 9.4-fold and that lowering translation rates reduced mRNA levels by up to 11.8-fold. We also found that RNAse binding sites in intergenic regions had much lower effects on mRNA levels. Surprisingly, changing transcriptional termination efficiency or introducing long single-stranded RNA into 3' UTRs had no effect on upstream mRNA levels. From these measurements, we developed and validated biophysical models of ribosome protection and RNAse activity with excellent quantitative correspondence. We also formulated design rules to rationally control a mRNA's stability, facilitating the automated design of engineered genetic systems with desired functionalities.

中文翻译：

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控制细菌操纵子中mRNA稳定性的序列和结构决定簇的系统定量

mRNA降解是影响所有基因表达水平的核心过程，但控制mRNA衰变速率的决定因素仍然难以确定。在这里，我们应用了一种合成的生物学设计学习方法来阐明控制细菌操纵子中mRNA稳定性的序列和结构决定簇。我们设计，构建和表征了82个操纵子，系统地改变了5'UTR，基因间和3'UTR区域中的RNAse结合位点特征，翻译起始速率和转录终止子效率，并使用RT-qPCR分析法测量了其mRNA水平。我们表明，将长单链RNA引入5'UTRs可以将mRNA水平降低多达9.4倍，降低翻译率可以将mRNA水平降低高达11.8倍。我们还发现，基因间区域中的RNA酶结合位点对mRNA水平的影响要低得多。出人意料的是，改变转录终止效率或将长单链RNA引入3'UTRs对上游mRNA水平没有影响。通过这些测量，我们开发并验证了核糖体保护和RNAse活性的生物物理模型，并具有良好的定量对应关系。我们还制定了设计规则，以合理控制mRNA的稳定性，从而促进具有所需功能的基因工程系统的自动化设计。我们开发并验证了具有良好定量对应关系的核糖体保护和RNAse活性的生物物理模型。我们还制定了设计规则以合理地控制mRNA的稳定性，从而促进了具有所需功能的基因工程系统的自动化设计。我们开发并验证了具有良好定量对应关系的核糖体保护和RNAse活性的生物物理模型。我们还制定了设计规则以合理地控制mRNA的稳定性，从而促进了具有所需功能的基因工程系统的自动化设计。