Measuring the tolerance of the genetic code to altered codon size

Area of Science:

• Molecular Biology
• Synthetic Biology
• Genetics

Background:

• The genetic code uses three-base codons for translation.
• Understanding constraints on codon size is crucial for genetic code evolution and engineering.

Purpose of the Study:

• To investigate the feasibility and limitations of converting the Escherichia coli genetic code to utilize four-base codons.
• To identify constraints imposed by transfer RNA (tRNA) and aminoacyl-tRNA synthetase (aaRS) compatibility with expanded codon sizes.

Main Methods:

• Engineering of transfer RNAs (tRNAs) from all 20 canonical isoacceptor classes to recognize four-base codons (qtRNAs).
• Mass spectrometry to confirm selective amino acid incorporation by engineered qtRNAs.
• Assessing the compatibility of aminoacyl-tRNA synthetases (aaRSs) with quadruplet anticodons.

Main Results:

• All 20 canonical isoacceptor tRNAs can be converted to functional quadruplet tRNAs (qtRNAs).
• Many qtRNAs selectively incorporate a single amino acid for a specific four-base codon.
• Efficient quadruplet codon translation often requires multiple tRNA mutations, and only nine out of twenty aaRSs tolerate quadruplet anticodons, limiting the available chemical alphabet.

Conclusions:

• A four-base codon system is achievable but presents significant biochemical constraints compared to the triplet codon system.
• The universal triplet codon code was likely selected for its simplicity and sufficiency, not due to the inherent unachievability of a quadruplet code.
• The findings provide a foundation for synthetic biologists aiming to engineer novel, expanded genetic codes with four-base codons.