Kinetics of d-Amino Acid Incorporation in Translation.

Area of Science:

• Biochemistry
• Molecular Biology
• Synthetic Biology

Background:

• Ribosomal translation typically uses only l-amino acids (l-AAs).
• Synthetic biology enables in vitro incorporation of d-amino acids (d-AAs) for novel polypeptide properties.
• The precise steps limiting d-AA incorporation during translation remain unclear.

Purpose of the Study:

• To elucidate the rate-limiting steps for d-amino acid incorporation during ribosomal translation.
• To compare the efficiency of d- and l-amino acid incorporation using kinetic analysis.

Main Methods:

• Utilized quench-flow kinetics to measure amino acid incorporation rates.
• Employed tRNA engineering (tRNA body swaps) and varied elongation factor Tu (EF-Tu) concentrations.
• Assessed d- and l-phenylalanine (Phe) incorporation using specific tRNA adaptors (tRNA^PheB and tRNA^AlaB).

Main Results:

• d-Phe incorporation into dipeptides was 250-fold slower than l-Phe from tRNA^PheB.
• Higher EF-Tu concentrations and tRNA modifications partially accelerated d-AA incorporation, suggesting EF-Tu binding can be rate-limiting.
• Despite slow incorporation, d-Phe competed efficiently with l-Phe from tRNA^AlaB at saturating EF-Tu, indicating rapid EF-Tu and ribosome binding.
• Accommodation and peptide bond formation were identified as the primary bottlenecks for d-AA incorporation.
• Subsequent elongation with l-AAs after d-AA incorporation was significantly impaired.

Conclusions:

• Identified peptide bond formation as the key rate-limiting step for d-AA incorporation in vitro.
• Provided mechanistic insights into the stereospecificity of translation and factors influencing d-AA incorporation efficiency.
• Suggests strategies for optimizing d-AA incorporation in synthetic biological applications.