Metabolic stress promotes stop-codon readthrough and phenotypic heterogeneity.

Area of Science:

• Molecular Biology
• Microbiology
• Biochemistry

Background:

• Accurate protein synthesis relies on quality control by aminoacyl-tRNA synthetases and ribosomes.
• Stop-codon readthrough, where near-cognate aminoacyl-tRNAs suppress stop codons, leads to protein variants and cellular heterogeneity.
• Environmental factors influencing translational accuracy and stop-codon readthrough remain largely unknown.

Purpose of the Study:

• To investigate the impact of metabolic stress, specifically excess carbon, on the level and heterogeneity of stop-codon readthrough in bacterial cells.
• To elucidate the molecular mechanisms by which excess carbon influences translational accuracy.
• To assess the adaptive significance of increased stop-codon readthrough under stress conditions.

Main Methods:

• Dual-fluorescence reporters to quantify readthrough.
• Mass spectrometry to identify protein variants.
• Mathematical modeling to analyze readthrough dynamics.
• Single-cell approaches, including time-lapse microscopy, to study cellular heterogeneity and adaptation.

Main Results:

• Metabolic stress induced by excess carbon significantly increases both the level and heterogeneity of stop-codon readthrough.
• Accumulation of acid metabolites lowers pH, reducing release factor activity and promoting readthrough.
• Single cells exhibiting high readthrough levels demonstrate enhanced adaptation to severe acid stress and increased sensitivity to aminoglycoside antibiotics.

Conclusions:

• Excess carbon acts as a metabolic stress that promotes translational heterogeneity through altered release factor activity.
• Increased stop-codon readthrough contributes to phenotypic diversity and cellular adaptation to challenging environments.
• Translational accuracy is a dynamic process influenced by metabolic state, impacting bacterial survival and response to antibiotics.