Aminoacyl-tRNA Synthetases and tRNAs for an Expanded Genetic Code: What Makes them Orthogonal?

Abstract

In the past two decades, tRNA molecules and their corresponding aminoacyl-tRNA synthetases (aaRS) have been extensively used in synthetic biology to genetically encode post-translationally modified and unnatural amino acids. In this review, we briefly examine one fundamental requirement for the successful application of tRNA/aaRS pairs for expanding the genetic code. This requirement is known as "orthogonality"-the ability of a tRNA and its corresponding aaRS to interact exclusively with each other and avoid cross-reactions with additional types of tRNAs and aaRSs in a given organism.

Keywords: aminoacyl-tRNA synthetases; expanded genetic code; orthogonal translation systems; synthetic biology; tRNA.

Figure 1

tRNA molecules and corresponding aminoacyl-tRNAs that have been used to expand the genetic code of live cells. Crystal structures of tRNA molecules (in white) are shown in complex with the corresponding aminoacyl-tRNAs (in the colors of the rainbow). The labels next to each structure indicate the origin of the orthogonal tRNA/tRNA synthetase pair and an organism into which this pair has been transplanted, which is followed by a brief description of tRNA identity or anti-identity elements that make a tRNA/tRNA synthetase pair orthogonal in an organism into which this pair has been transplanted. For example, the label “TyrRS-tRNA^Tyr—From S. cerevisiae to E. coli: G1-C72 in bacteria vs C1-G72 in eukaryotes” means that the TyrRS-tRNA^Tyr pair has been transplanted from S. cerevisiae into E. coli where this pair remains orthogonal due to different identity elements between bacterial and eukaryotic tRNA^Tyr: the G1-C72 pair in bacterial tRNA^Tyr and the C1-G72 pair in eukaryotic tRNA^Tyr. The asterisk next to the LysRS-tRNA^Lys complex indicates that this model was produced by docking the crystal structure of tRNA^Lys into the tRNA-binding pocket of LysRS. The most comprehensive list of orthogonal tRNA/tRNA synthetase pairs and their classes can be found in the Supplemental files of Ref. [1].