The incorporation of non-canonical amino acids (ncAAs) is only possible if the translational mechanisms are adapted. One possible way to incorporate the ncAAs is the repurposing of codons like the amber stop or rarely used leucine codons. Another way would be the incorporation of an unnatural base into the DNA to get new codons. For both, an orthogonal tRNA/aminoacyl-synthetase is necessary, which could charge the ncAA to the tRNA.
Adaption of the Translational Mechanism to Incorporate Non-canonical Amino Acids
By the incorporation of the unnatural base pairs (UBP), the translational system enables the recognition of the new bases and the interaction of a matching tRNA/synthetase pair. This is the starting point for the engineering of an orthogonal tRNA/synthetase pair for the translational incorporation of certain non-canonical amino acids (ncAA). In the process of protein biosynthesis, the DNA is first transcribed into mRNA and in the next step into amino acid polypeptides. Therefore, the ribosome binds on a certain position for the mRNA and supports the binding of the interaction for the mRNA and the matching amino acyl tRNA. The ribosome contains three binding sites. The tRNA first binds in the A site and the peptidyl tRNA is bound in the P-site. The polypeptide is then transferred from the peptidyl tRNA in the P-site to the aminoacyl-tRNA in the A-site. In the process of translocation, the ribosome moves on to the next codon on the mRNA and the tRNA is aminoacylated by the tRNA synthetase (aaRS). The tRNA leaves the complex of mRNA and ribosome through the E-site. Thus, the polypeptide chain is extended in numerous of these processes and the polypeptide is formed. This complex mechanism had to be adapted due to the use of UBPs.
The first step of the two-step reaction is the formation of an aminoacylated intermediate by the reaction of the amino acid and ATP. This is possible through the Rossmann fold, a domain consiting of a six-stranded parallel β-sheet with connecting helices. The Rossmann fold acts analog to the active site of hydrogenases and is responsible for binding ATP, the amino acid, and the 3’-terminus of the tRNA.
The class I synthetases are monomeric synthetases and feature all structurally similar active site Rossmann fold domains in the region of the N-terminus. Beside this region, there are no significant structural or sequence similarities among the class I enzymes. There is an acceptor binding site inserted into the Rossmann-fold domain at a common location which binds the single stranded terminal end of the tRNA, while its C-terminal domain binds in the minor groove of the L-shaped tRNA and the anticodon arm. That is the point for the discrimination among the different tRNAs. The binding requires the formation of a hairpin structure of the single stranded 3’-terminus with the amino acid and the ATP in the active site (Chang et al., 2010, Krebs et al., 2014).
Figure 1: Crystal structure of a class I tRNA/aminoacyl synthetase.
The tRNA is shown in red and the protein in blue (Krebs et al., 2014).
An Orthogonal tRNA/Aminoacyl-Synthetase Pair
Since the tRNA recognition by the aaRS can be domain or species specific (Kwok et al., 1980), a heterologous aaRS/tRNA pair from a different organism is used. A possible source for an orthogonal tRNA/synthetase pair for the application in bacterial cells are eukaryotes. However, the adaption of these tRNA/aaRS pair which aminoacylates in E. coli is very difficult (Liu et al., 1999). Evolved synthetases from archaea can be expressed efficiently in E. coli (Wang et al., 2001, Wang et al., 2000) and at the same time are more similar to eukaryotic organisms than to the prokaryotic host (Kwok et al., 1980, Zhang et al., 2005). The reason are significant differences in the acceptor stem and anticodon binding recognition domain between prokaryotic and archaea tRNA/aaRS (Wang et al., 2000).
One possible orthogonal tRNA/aaRS pair is the tyrosine tRNA/aaRS pair of Methanococcus jannaschii, where the first base pair of the acceptor stem CG as a tRNATyridentity element differs from those of E. coli tRNATyr containing a GC (Wang et al., 2001). The orthogonal tRNA/aaRS pair is incorporated as a response of the amber stop codon, which does not incorporate any of the 20 canonical amino acids. We use the amber stop codon (UAG), because this codon is rare in E.coli and incorporation of random amino acids has been shown before.
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