Fidelity of protein synthesis in vivo
by Nicola Sonja Freyer
Date of Examination:2025-09-29
Date of issue:2025-11-12
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Markus Bohnsack
Persistent Address:
http://dx.doi.org/10.53846/goediss-11620
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Abstract
English
Protein synthesis fidelity is essential for maintaining proteome integrity and cellular fitness. Errors, such as amino acid misincorporations caused by incorrect decoding, result in aberrant proteins that promote proteotoxic stress. Aminoglycoside antibiotics (AGAs) target the ribosome and impair translational fidelity, ultimately disrupting proteostasis. However, key aspects of AGA action – including drug uptake dynamics, the quantitative thresholds of translation errors that trigger specific cellular responses, and the events resulting in proteostasis breakdown – remain poorly understood. Similarly, bacterial resistance mechanisms against AGAs are not fully elucidated. Using mass spectrometry-based approaches, we investigated AGA action in Escherichia coli. Global proteomic changes upon AGA treatment were quantified using data-independent acquisition (DIA). To quantify ribosomal misreading, we developed a pipeline that combines data-dependent acquisition (DDA) for unbiased detection of missense peptides with targeted mass spectrometry using parallel reaction monitoring (PRM) for validation and quantification. This enabled the identification of over 1,000 unique missense errors across 322 proteins, yielding the most comprehensive AGA-induced misreading profile to date and enabling quantification of rare errors at frequencies as low as 10⁻⁶. To dissect steady-state error levels in full-length proteins shaped by biosynthetic and quality control pathways, we adapted the PUNCH-P protocol to isolate ribosome-nascent chain complexes and enrich nascent peptides for mass spectrometric analysis. This analysis revealed that AGA uptake kinetics drive dynamic changes in the error landscape and that certain proteins – particularly stress-induced chaperones – show exceptionally high error rates due to synthesis under extreme misreading conditions. Cells respond by activating stress responses and metabolic reprogramming to prioritize survival. At bactericidal AGA concentrations, membrane disruption causes massive drug influx and translational arrest. Error frequencies in nascent chains reach ~10%, while full-length protein synthesis is severely impaired, leading to proteostasis collapse and cell death. In response to AGA treatment, bacteria preferentially evolve resistance mutations in the fusA gene, encoding elongation factor G. Our collaborative work suggests that fusA mutations selectively silence AGA-corrupted ribosomes, representing the first example of a new resistance strategy. We showed that fusA mutant strains retain low misreading upon AGA exposure, including in the inner membrane proteome. This preserves proteome and membrane integrity at minimal fitness costs, demonstrating that fusA mutations confer resistance early in the AGA uptake cycle. Overall, this work advances understanding of decoding fidelity under antibiotic pressure, uncovers a resistance mechanism based on selective ribosome silencing, and presents refined proteomic tools to query translational accuracy at scale.
Keywords: ribosome; antibiotic resistance; mass spectrometry; translational fidelity
