Pyrrolysyl-tRNA Synthetase with a Unique Architecture Enhances the Availability of Lysine Derivatives in Synthetic Genetic Codes

Abstract

Genetic code expansion has largely relied on two types of the tRNA—aminoacyl-tRNA synthetase pairs. One involves pyrrolysyl-tRNA synthetase (PylRS), which is used to incorporate various lysine derivatives into proteins. The widely used PylRS from Methanosarcinaceae comprises two distinct domains while the bacterial molecules consist of two separate polypeptides. The recently identified PylRS from Candidatus Methanomethylophilus alvus (CMaPylRS) is a single-domain, one-polypeptide enzyme that belongs to a third category. In the present study, we showed that the PylRS—tRNAPyl pair from C. M. alvus can incorporate lysine derivatives much more efficiently (up to 14-times) than Methanosarcinaceae PylRSs in Escherichia coli cell-based and cell-free systems. Then we investigated the tRNA and amino-acid recognition by CMaPylRS. The cognate tRNAPyl has two structural idiosyncrasies: no connecting nucleotide between the acceptor and D stems and an additional nucleotide in the anticodon stem and it was found that these features are hardly recognized by CMaPylRS. Lastly, the Tyr126Ala and Met129Leu substitutions at the amino-acid binding pocket were shown to allow CMaPylRS to recognize various derivatives of the bulky Nε-benzyloxycarbonyl-l-lysine (ZLys). With the high incorporation efficiency and the amenability to engineering, CMaPylRS would enhance the availability of lysine derivatives in expanded codes.

Naturally Occurring tRNAs With Non-canonical Structures

“…On the surface, the break in the base pairing of the anticodon stem as well as the lack of a connecting base between the acceptor and D-stem profile as potential identity elements for Ma tRNA Pyl . Interestingly, deletion of the unpaired nucleotide in the anticodon stem did not significantly alter the translation efficiency of Ma PylRS-tRNA Pyl in a cellfree translation system (Yamaguchi et al, 2018). Insertion of a C or U between the acceptor and D-stem (position 8) moderately decreased translation, but inserting an A or G had no effect (Yamaguchi et al, 2018).…”

“…Interestingly, deletion of the unpaired nucleotide in the anticodon stem did not significantly alter the translation efficiency of Ma PylRS-tRNA Pyl in a cellfree translation system (Yamaguchi et al, 2018). Insertion of a C or U between the acceptor and D-stem (position 8) moderately decreased translation, but inserting an A or G had no effect (Yamaguchi et al, 2018). This indicates that the absence of a base in this position may not be an identity element for Ma tRNA Pyl .…”

“…However, Ma PylRS is highly active toward its cognate tRNA Pyl even though it does not feature PylSn. Despite significant structural differences between Ma and Mm tRNA Pyl , Ma tRNA Pyl can serve as a substrate for both PylRS enzymes (Yamaguchi et al, 2018). However, lengthening the variable arm of Ma tRNA Pyl prevents aminoacylation by Mm PylRS, due to steric constraints between PylSn and the enlarged variable arm as discussed earlier (Suzuki et al, 2017).…”

“…Ultimately, Dh PylSc binds to tRNA Pyl through contacts with the acceptor and D-stem, and has no direct contact with the anticodon stem, variable loop, or T-stem (Nozawa et al, 2009). The PylRS-tRNA Pyl pair in the seventh-order methanogen M. alvus has recently been explored as an additional tool for genetic code expansion with advantages over its previously studied counterparts (Meineke et al, 2018;Willis and Chin, 2018;Yamaguchi et al, 2018;Beránek et al, 2019;Dunkelmann et al, 2020;Seki et al, 2020). Ma tRNA Pyl has many unusual features that distinguish it from canonical tRNAs as well as previously characterized tRNA Pyl (Figure 8C).…”

Synthetic Tyrosine tRNA Molecules with Noncanonical Secondary Structures

“…Dh tRNA Pyl assumes a similar secondary structure to that of Mm tRNA Pyl , whereas CM a tRNA Pyl has such idiosyncrasies as no nucleotide between the acceptor and D stems, a 4-nucleotide D loop, and an extra nucleotide in the 6-base pair anticodon stem [4]. Since the removal of this extra residue does not impair the in vivo tRNA activity [18], we used the sequence of this deletion variant of CMa tRNA Pyl for the transplantation. PYLYCMa showed a higher suppression activity than PYLY1 and PYLY2, while PYLYDh was also aminoacylated by Mj TyrRS, although its suppressor activity was lower than that of PYLY1 (Table 1 and Figure S1B).…”