Absolute quantification of translational regulation and burden using combined sequencing approaches
Absolute quantification of translational regulation and burden using combined sequencing approaches
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DOI：
10.15252/msb.20188719
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发表时间：
2019-05-01
影响因子：
9.9
通讯作者：
Ignatova, Zoya
中科院分区：
文献类型：
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作者：
Gorochowski, Thomas E.;Chelysheva, Irina;Ignatova, Zoya
mrna翻译成蛋白质是一个关键的细胞过程。核糖体结合位点和终止密码子提供启动和终止翻译的信号，而稳定的次级mRNA结构可以诱导翻译重编码事件。荧光蛋白通常用于表征这些元素，但需要修改部分的自然环境，并且只允许同时监测少数参数。在这里，我们将核糖核酸测序与定量rna测序结合起来，以核苷酸分辨率和绝对单位测量蛋白质合成过程中控制转录和翻译过程的元件的性能。我们同时测量了大肠杆菌转录组的779个翻译起始率和750个翻译终止效率，以及在稳定的RNA假结结构下诱导的翻译移框。通过分析转录和翻译反应，我们发现假结处隔离的核糖体有助于sigma（32）介导的应激反应、密码子特异性暂停和整个细胞翻译起始率的下降。我们的工作证明了整合全球方法对活细胞中基因调控和负担的全面定量理解的力量。
Translation of mRNAs into proteins is a key cellular process. Ribosome binding sites and stop codons provide signals to initiate and terminate translation, while stable secondary mRNA structures can induce translational recoding events. Fluorescent proteins are commonly used to characterize such elements but require the modification of a part's natural context and allow only a few parameters to be monitored concurrently. Here, we combine Ribo-seq with quantitative RNA-seq to measure at nucleotide resolution and in absolute units the performance of elements controlling transcriptional and translational processes during protein synthesis. We simultaneously measure 779 translation initiation rates and 750 translation termination efficiencies across the Escherichia coli transcriptome, in addition to translational frameshifting induced at a stable RNA pseudoknot structure. By analyzing the transcriptional and translational response, we discover that sequestered ribosomes at the pseudoknot contribute to a sigma(32)-mediated stress response, codon-specific pausing, and a drop in translation initiation rates across the cell. Our work demonstrates the power of integrating global approaches toward a comprehensive and quantitative understanding of gene regulation and burden in living cells.