Environment Modulates Protein Heterogeneity Through Transcriptional and Translational Stop Codon Miscoding

Abstract

ABSTRACTStop codon miscoding events give rise to longer proteins, which may alter the protein’s function and thereby generate short-lasting phenotypic variability from a single gene.In order to systematically assess the frequency and origin of stop codon miscoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors inE.coliusing fluorescent microscopy. We found that under stress conditions, stop codon miscoding may occur with a rate as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon miscoding 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 miscoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon miscoding by mass spectrometry revealed that temperature regulates the expression of cryptic sequences generated by stop codon miscoding inE.coli.Overall, our findings suggest that the environment influences the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.