期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2020
卷号：117
期号：36
页码：22167-22172
DOI：10.1073/pnas.2013543117
出版社：The National Academy of Sciences of the United States of America
摘要：Accurate protein synthesis is a tightly controlled biological process with multiple quality control steps safeguarded by aminoacyl-transfer RNA (tRNA) synthetases and the ribosome. Reduced translational accuracy leads to various physiological changes in both prokaryotes and eukaryotes. Termination of translation is signaled by stop codons and catalyzed by release factors. Occasionally, stop codons can be suppressed by near-cognate aminoacyl-tRNAs, resulting in protein variants with extended C termini. We have recently shown that stop-codon readthrough is heterogeneous among single bacterial cells. However, little is known about how environmental factors affect the level and heterogeneity of stop-codon readthrough. In this study, we have combined dual-fluorescence reporters, mass spectrometry, mathematical modeling, and single-cell approaches to demonstrate that a metabolic stress caused by excess carbon substantially increases both the level and heterogeneity of stop-codon readthrough. Excess carbon leads to accumulation of acid metabolites, which lower the pH and the activity of release factors to promote readthrough. Furthermore, our time-lapse microscopy experiments show that single cells with high readthrough levels are more adapted to severe acid stress conditions and are more sensitive to an aminoglycoside antibiotic. Our work thus reveals a metabolic stress that promotes translational heterogeneity and phenotypic diversity. Faithful gene expression demands high fidelity during DNA replication, transcription, and translation ( 1 ⇓ ⇓ ⇓ ⇓ ⇓ – 7 ). A critical step to maintain such accuracy is proper termination of protein synthesis at UAA, UAG, and UGA stop codons, which is catalyzed by release factors (RFs) ( 8 , 9 ). In bacteria, RF1 catalyzes release of peptides from the peptidyl-transfer RNA (tRNA) at UAA and UAG codons, whereas RF2 releases growing peptides at UAA and UGA codons. In eukaryotes, a single class I release factor, eRF1, terminates translation at all three stop codons on the ribosome. In nature and in the laboratory, stop codons can be read through with high efficiency by suppressor tRNAs, which carry mutations in the anticodon to match the stop codons ( 10 ). This approach is widely used to site-specifically insert noncanonical amino acids into proteins of interest ( 11 , 12 ). Even in cells lacking cognate suppressor tRNA, readthrough of stop codons is still prevalent, albeit at lower rates. A global analysis of translation termination in Escherichia coli with ribosome profiling reveals that ribosomes occupy the messenger RNA (mRNA) region following the stop codons of hundreds of genes ( 13 ). Proteomic analyses also demonstrate that near-cognate aminoacyl-tRNAs are able to read stop codons ( 14 , 15 ). For example, glutamine and tryptophan suppress UAG and UGA codons, respectively. In addition, readthrough of stop codons are programmed events in multiple mammalian and viral genes ( 16 , 17 ). Stop-codon readthrough adds an extended amino acid sequence to the C terminus and has been shown to be critical for expression of functional protein variants. For example, readthrough of a UAG stop codon in gag is critical for expression of a protein variant and assembly of retrovirus ( 17 ). Stop-codon readthrough also yields a functional peroxisomal lactate dehydrogenase in mammals ( 18 ) and alters protein localization in fungi and animals ( 19 , 20 ). Furthermore, targeting readthrough of premature stop codons has been actively explored as a therapeutic strategy to treat genetic diseases ( 21 , 22 ). In bacteria, readthrough of stop codons regulates the expression levels of RF2 and amino acid biosynthesis genes ( 13 , 23 , 24 ). Despite these well-established examples of specific functional readthrough products, how global stop-codon readthrough alters during environmental shifts and affects cell fitness remains largely unknown. We have previously developed dual-fluorescence reporters to conveniently quantitate stop-codon readthrough levels in the population and single bacterial cells ( 15 ). In a screen for growth conditions that affect stop-codon readthrough using these reporters in E. coli , we have uncovered that excess carbon in media substantially increases the readthrough levels of UAA, UAG, and UGA codons. We further show that such an increase depends on a drop of pH during bacterial growth in excess carbon. Interestingly, excess carbon and low pH not only increase the average level of readthrough, but also enhance its fluctuation among single bacterial cells. The individual cells with low and high readthrough levels are better prepared for distinct stress conditions, suggesting that the metabolic stress caused by excess carbon promotes heterogeneity of stop-codon readthrough and phenotypic diversity. Results Excess Carbon Promotes Stop-Codon Readthrough. To date, little is known about how environmental factors affect stop-codon readthrough. To address this question, we used our previously developed dual-fluorescence reporter system ( 15 ) to screen the UGA readthrough level of wild-type (WT) E. coli (MG1655) grown in various media ( SI Appendix , Fig. S1 ). Previous studies have indicated that carbon starvation increases the level of stop-codon readthrough ( 25 ), although the underlying mechanism remains unclear. Unexpectedly, we found that adding extra carbon sources (e.g., glucose) into the rich medium Luria–Bertani broth (LB) also significantly increased the level of UGA readthrough ( SI Appendix , Fig. S1 ). We next tested the effect of excess glucose on stop-codon readthrough and frameshift in both the WT and error-prone ( rpsD* ) strains. The rpsD* strain carries an I199N mutation in the ribosomal small subunit rpsD gene and displays increased errors during ribosomal decoding ( 26 , 27 ). Addition of 1% glucose in LB increased the level of UGA and UAA readthrough three- to seven-fold in both MG1655 and rpsD* ( Fig. 1 A ). Although UAG readthrough is not detected with the dual-fluorescence reporter in MG1655, excess glucose clearly increased UAG readthrough in the rpsD* mutant. In contrast, little change was observed for frameshift upon addition of glucose, suggesting that the effect of excess carbon on ribosomal fidelity is specific for stop-codon readthrough.
