ENVIRONMENT MODULATES PROTEIN HETEROGENEITY THROUGH TRANSCRIPTIONAL AND TRANSLATIONAL STOP CODON RECODING
In order to systematically assess the frequency and origin of stop codon recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur with a rate as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon recoding 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 recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon recoding by mass spectrometry revealed that temperature regulates the expression of cryptic sequences generated by stop codon recoding in E. 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. Overall design: We designed several reporters for stop codon recoding events. The strategy dwells on introducing a premature stop codon into an mScarlet allele. Thus, only upon stop codon recoding, full-length and, therefore, functional mScarlet will be synthesized. Here, we studied the mRNA sequence of 12 reporters for stop codon recoding events and the mScarlet wild-type, expressed in wild-type E. coli grown at 18? in LB media. The reporters for stop codon recoding were ordered from Twist Biosciences.