Synthetic Tyrosine tRNA Molecules with Noncanonical Secondary Structures

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• Transfer RNA (tRNA) structure is crucial for its activity, with tertiary interactions in the core maintaining the L-shape.
• Alterations in the tRNA core can lead to structural defects and reduced function.

Purpose of the Study:

• To develop a method for safely engineering structural variations in the tRNA core.
• To utilize the noncanonical scaffold of tRNA^Pyl for creating novel tRNA structures.
• To explore the potential of engineered tRNAs for genetic code reprogramming.

Main Methods:

• Construction of a hybrid tRNA combining elements of archaeal tRNA^Pyl and tRNA^Tyr.
• Testing the hybrid tRNA's ability to translate the UAG codon to 3-iodotyrosine in *Escherichia coli*.
• Systematic modification of the hybrid tRNA to generate artificial tRNA variants (ZtRNA and YtRNA).

Main Results:

• The initial hybrid tRNA successfully translated the UAG codon to 3-iodotyrosine, albeit with slightly lower efficiency than a benchmark tRNA.
• Two artificial tRNA^Tyr variants, ZtRNA and YtRNA, were created with distinct structural modifications.
• ZtRNA featured a noncanonical augmented D (auD) helix and standard D/T loop interactions, while YtRNA had a canonical auD helix and non-standard interloop interactions.

Conclusions:

• The engineered tRNA scaffolds, particularly ZtRNA, demonstrate potential for supporting various aminoacylations, including glycylation and glutaminylation.
• Synthetic diversity in tRNA engineering can facilitate the creation of new tRNA-aminoacyl-tRNA synthetase pairs.
• This approach holds promise for expanding the repertoire of amino acids incorporated into proteins, thereby reprogramming the genetic code.