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Review
. 2018;15(4-5):453-460.
doi: 10.1080/15476286.2017.1343227. Epub 2017 Sep 6.

tRNA engineering for manipulating genetic code

Takayuki Katoh  1   2 , Yoshihiko Iwane  1 , Hiroaki Suga  1   3
Affiliations
Review

tRNA engineering for manipulating genetic code

Takayuki Katoh et al. RNA Biol. 2018.

Abstract

In ribosomal translation, only 20 kinds of proteinogenic amino acids (pAAs), namely 19 l-amino acids and glycine, are exclusively incorporated into polypeptide chain. To overcome this limitation, various methods to introduce non-proteinogenic amino acids (npAAs) other than the 20 pAAs have been developed to date. However, the repertoire of amino acids that can be simultaneously introduced is still limited. Moreover, the efficiency of npAA incorporation is not always sufficient depending on their structures. Fidelity of translation is sometimes low due to misincorporation of competing pAAs and/or undesired translation termination. Here, we provide an overview of efforts to solve these issues, focusing on the engineering of tRNAs.

Keywords: aminoacyl-tRNA synthetase; flexizyme; genetic code reprogramming; non-proteinogenic amino acid; peptide; ribosome; tRNA; translation.

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Figures

Figure 1.
Figure 1.
Development of orthogonal tRNAs. (A) Conceptual scheme of orthogonal ARS/tRNA pairs. ARS1 specifically charges amino acid 1 (AA1) onto tRNA1, but not onto tRNA2. Conversely, tRNA2 is exclusively charged with amino acid 2 (AA2) by ARS2, but not with AA1 by ARS1. (B) Mutant TyrRS/tRNATyr CUA pair orthogonal to the E. coli wild-type (WT) TyrRS/tRNATyr pair. The mutant TyrRS was developed based on M. jannaschii TyrRS to charge O-methyltyrosine instead of tyrosine on M. jannaschii tRNATyr or mutRNATyr CUA. The mutRNATyr CUA has 5 nucleotide substitutions, C17A, U17aG, U20C, G37A, and U47G, to improve the orthogonality as well as the anticodon substitution from GUA to CUA to decode UAG codon. (C) Methanococcus maripaludis (Mm) SepRS/tRNASep CUA pair orthogonal to the E. coli wild-type (WT) CysRS/tRNACys pair. The tRNASep CUA was designed based on M. jannaschii tRNACys with 3 nucleotide changes (C20U, G34C, and C35U). (D) Secondary structure of tRNAAsnE2 GGA designed based on E. coli tRNAAsn. Nucleotide changes at the acceptor stem are introduced to give orthogonality to the other E. coli ARS/tRNA pairs. (E) Secondary structure of tRNAGluE2 GGA designed based on E. coli tRNAGlu.
Figure 2.
Figure 2.
Overview of orthogonal ribosome/tRNA pairs. The mutant ribosome/tRNA pair consists of the mutant ribosome with G2251C/G2253C mutations in the 23S rRNA and the tRNA with C75G mutation. The mutant ribosome specifically recognizes the mutant tRNA, and is inert to the wild-type (WT) tRNA. Similarly, the WT ribosome exclusively utilizes the WT tRNA. These coexisting orthogonal machineries express 2 distinct peptides from a single mRNA template depending on the artificially reprogrammed genetic code and the WT genetic code.
Figure 3.
Figure 3.
Schematic representation of the artificial division of codon boxes. (A) The genetic code of a reconstituted translation system containing E. coli native tRNA mixture. The GUN codons in valine codon box are decoded by 2 kinds of native tRNAVals. (B) A reprogrammed genetic code where 32 in vitro transcribed tRNAs decodes 31 NNS (S = C or G) elongation codons along with the AUG initiation codon. (C) A reprogrammed genetic code containing 23 building blocks by means of artificial division of 3 codon boxes. The 3 nonproteinogenic amino acids (npAAs) are assigned to the black background codons by replacing the redundant tRNAVal GAC, tRNAArg GCG and tRNAGly GCC with the 3 bioorthogonal npAA-tRNAGNN's prepared by flexizyme-mediated aminoacylation.
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