Mistakes in translation: Reflections on mechanism

Area of Science:

• Molecular Biology
• Biochemistry
• Protein Synthesis

Background:

• Errors in messenger RNA (mRNA) translation into protein can impact recombinant protein production for biophysical and biopharmaceutical applications.
• These translation errors, often involving substitutions between amino acids coded by codons differing in a single base (e.g., lysine AAA to arginine AGA), can offer insights into the translation mechanism.

Purpose of the Study:

• To investigate the frequency variation of specific amino acid mis-incorporations (lysine for arginine) at different sites within a recombinant protein.
• To explore the potential influence of codon context, such as clustering of rare codons, on the fidelity of translation.

Main Methods:

• Utilized mass spectrometry for sensitive detection of amino acid mis-incorporations.
• Employed parallel expression of wild-type and codon-optimized genes in differentially isotope-enriched media (15N and 14N) to quantify mis-incorporation frequencies.

Main Results:

• Demonstrated significant variation in lysine for arginine mis-incorporation frequencies (0-16%) across nine distinct sites containing a rare arginine codon in yeast ADP-ribosylation factor expressed in E. coli.
• Observed that mis-incorporation frequencies were not uniform, contradicting expectations based solely on tRNA abundance.

Conclusions:

• Codon context, particularly the clustering of rare codons, is proposed as a key factor influencing the site-specific frequency of amino acid mis-incorporations during translation.
• The developed mass spectrometry-based method offers a sensitive and efficient approach for studying translation errors, with potential applications in refining models of ribosomal translation elongation.