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198282 https://hdl.handle.net/2286/R.2.N.198282 http://rightsstatements.org/vocab/InC/1.0/ All Rights Reserved 2024 194 pages Doctoral Dissertation Academic theses Text eng Stikeleather, Ryan Lynch, Michael Hansen, Debra Frasch, Wayne Arizona State University Partial requirement for: Ph.D., Arizona State University, 2024 Field of study: Biological Design Translation is a fundamental process of life, yet methods to systematically investigate the fidelity of it have been limited. Historically, mistranslation has mostly been approached from the vantage of considering a single codon at a time in a single protein. Here, a method is presented that enables the investigation of nearly all pairwise amino-acid substitutions, thereby revealing both the rate and spectra of mistranslation events per amino acid. This approach also extends to evaluating fidelity at the codon level. The method is amenable for use with existing mass-spectrometry data. This investigation focuses on historically characterized ribosomal variants of Escherichia coli. While determining error rates is illuminating, understanding why certain mistranslation events are enriched requires further exploration, e.g., the relevance to the translation-accuracy hypothesis. It was found that preferred codons are not predictive of translational fidelity. Protein abundance was instead a better predictor of translation error, with an observed positive correlation between abundance and translation error rate. Moreover, the historically characterized error-prone ribosomal variant of E. coli was found to have a mistranslation bias that would confound the results of prior reporter assays that utilized the strain. The ribosomal variants each exhibited unique mistranslation profiles, differing in the types of translation-error events experienced. However, there was no significant difference among the overall translation error rates of the ribosomal variants, illustrating the importance of a method that enables proteome-wide translational accuracy studies. Molecular Biology Bioinformatics Mass Spectrometry Protein Abundance Translation Translation Error Mass Spectrometry and Bioinformatics for Determining Translation Error Rates