Abstract
Transfer RNA (tRNA) is a dynamic molecule used by all forms of life as a key component of the translation apparatus. Each tRNA is highly processed, structured, and modified, to accurately deliver amino acids to the ribosome for protein synthesis. The tRNA molecule is a critical component in synthetic biology methods for the synthesis of proteins designed to contain non-canonical amino acids (ncAAs). The multiple interactions and maturation requirements of a tRNA pose engineering challenges, but also offer tunable features. Major advances in the field of genetic code expansion have repeatedly demonstrated the central importance of suppressor tRNAs for efficient incorporation of ncAAs. Here we review the current status of two fundamentally different translation systems (TSs), selenocysteine (Sec)- and pyrrolysine (Pyl)-TSs. Idiosyncratic requirements of each of these TSs mandate how their tRNAs are adapted and dictate the techniques used to select or identify the best synthetic variants.
Highlights
Genetic code expansion (GCE) involves the engineering of protein synthesis machinery to site- incorporate non-canonical amino acids into a desired protein [1,2]. This is routinely done by assigning the ncAA to recoded stop or sense codons and delivering the ncAA to the ribosome via a suppressor transfer RNA
The identity elements of transfer RNA (tRNA) are nucleotides and their modifications, which function as substrate recognition determinants
Nonsense suppression is the most common way to insert ncAAs, since recoding is less detrimental to the proteome, given the low occurrence of stop codons
Summary
Genetic code expansion (GCE) involves the engineering of protein synthesis machinery to site- incorporate non-canonical amino acids (ncAAs) into a desired protein [1,2]. This is routinely done by assigning the ncAA to recoded stop or sense codons and delivering the ncAA to the ribosome via a suppressor transfer RNA (tRNA). PylRStRNAPyl pairs are used to engineer proteins with unique properties and functions in bacteria, viruses, insects, yeast, and animals [7,8,9,10,11] Another valuable building block for protein engineering is the 21st amino acid, selenocysteine (Sec).
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