PDF(Pubmed)
Abstract：
Stop codon readthrough events give rise to longer proteins, which may alter the protein\'s function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.
摘要：
终止密码子连读事件会产生更长的蛋白质，这可能会改变蛋白质的功能，从而从单个基因产生短暂的表型变异性。为了系统地评估终止密码子连读事件的频率和起源，我们设计了一个记者图书馆。我们在mScarlet中引入了过早终止密码子，这使得能够使用荧光显微镜对大肠杆菌中的蛋白质合成终止错误进行高通量定量。我们发现在压力条件下，终止密码子连读率可能高达80%，根据核苷酸的背景，这表明进化样本经常会停止密码子通读事件。通过质谱和RNA-seq对选定的报道分子的分析表明，不仅翻译，而且转录错误也会导致终止密码子连读。RNA聚合酶更有可能在过早终止密码子处错误掺入核苷酸。通过质谱对终止密码子连读的全蛋白质组检测揭示,温度调节大肠杆菌中由终止密码子连读产生的隐蔽序列的表达。总的来说,我们的研究结果表明，环境影响蛋白质生产的准确性，当生物体需要适应新条件时，这会增加蛋白质的异质性。
