Decoding of tandem quadruplets by adjacent tRNAs with eight-base anticodon loops

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetics

Background:

• Expanding the genetic code requires unique codons for non-natural amino acids.
• Quadruplet codons are being investigated as a potential expansion strategy.
• Engineered transfer RNA (tRNA) with modified anticodon loops is crucial for decoding novel codons.

Purpose of the Study:

• To investigate the potential of quadruplet codons for expanding the genetic code.
• To determine the decoding efficiency of tandem UAGA quadruplets by an engineered tRNA(Leu).
• To explore the influence of upstream codons and release factors on translation of novel codons.

Main Methods:

• Engineering a tRNA(Leu) with an eight-base anticodon loop for quadruplet codon recognition.
• Utilizing tandem UAGA quadruplets as potential codons for non-natural amino acids.
• Partially inactivating release factor 1 to facilitate decoding of non-standard codons.
• Analyzing the influence of the 5' codon on the decoding of UAGA quadruplets.

Main Results:

• Tandem UAGA quadruplets, when preceded by GCC and with partially inactivated release factor 1, specified two leucines with at least 10% efficiency.
• A purine at anticodon loop position 32 of the tRNA's decoding codon 5' to the UAGA influenced subsequent codon translation.
• Observed intraribosomal dissociation and re-pairing of anticodons to mRNA at overlapping or nearby codons, impacting frameshifting mechanisms.

Conclusions:

• Engineered tRNA can decode quadruplet codons, demonstrating a viable strategy for genetic code expansion.
• Translation fidelity of novel codons is influenced by upstream sequences and ribosomal dynamics.
• The findings provide insights into programmed frameshifting mechanisms and the potential of non-standard codon usage.