Environment Modulates Protein Heterogeneity Through Transcriptional and Translational Stop Codon Miscoding

Abstract

Stop 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 in E.coli using 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 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.

Evidence of a Protein-Coding Gene Antisense to the UL5 Gene in Bovine Herpesvirus I

“…This transversion from cytosine to guanine residue could be part of the discrepancy between the transcript length and the protein product of ORF M. One plausible explanation would be that the stop codon is being misread by suppressor tRNAs. Suppressor tRNAs incorporate tyrosine, glutamine, leucine, cysteine, lysine, arginine, or tryptophan in the place of a stop codon depending on the organism and type of stop codon, as has been observed in viruses, prokaryotes, and eukaryotes [59][60][61][62]. The relative abundance of certain tRNAs, particularly tyrosine and tryptophan, has also been shown to influence translation efficiency in a tissue-specific manner, and increases in these tRNAs correlate with higher levels of stop codon readthrough (or SC-RT) [63].…”