Synthetic Tyrosine tRNA Molecules with Noncanonical Secondary Structures

Abstract

The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity. Here, we report on a method to safely engineer structural variations in this domain utilizing the noncanonical scaffold of tRNAPyl. First, we constructed a naïve hybrid between archaeal tRNAPyl and tRNATyr, which consisted of the acceptor and T stems of tRNATyr and the other parts of tRNAPyl. This hybrid tRNA efficiently translated the UAG codon to 3-iodotyrosine in Escherichia coli cells, when paired with a variant of the archaeal tyrosyl-tRNA synthetase. The amber suppression efficiency was slightly lower than that of the “bench-mark” archaeal tRNATyr suppressor assuming the canonical structure. After a series of modifications to this hybrid tRNA, we obtained two artificial types of tRNATyr: ZtRNA had an augmented D (auD) helix in a noncanonical form and the D and T loops bound by the standard tertiary base pairs, and YtRNA had a canonical auD helix and non-standard interloop interactions. It was then suggested that the ZtRNA scaffold could also support the glycylation and glutaminylation of tRNA. The synthetic diversity of tRNA would help create new tRNA–aminoacyl-tRNA synthetase pairs for reprogramming the genetic code.

Directed Evolution of the Methanosarcina barkeri Pyrrolysyl tRNA/aminoacyl tRNA Synthetase Pair for Rapid Evaluation of Sense Codon Reassignment Potential

“…The unusual features of both the Methanosarcina sp. pyrrolysyl tRNA and its cognate aaRS contribute to this pair’s broad organismal orthogonality [ 47 ]. The aaRS is different from other type II synthetases in that the tRNA-binding elements wrap around the tRNA and make very specific contacts with the tRNA body [ 48 ].…”

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

“…More sophisticated orthogonal tRNA/synthetase pairs were engineered by constructing chimeric tRNAs [22] or by focused tRNA mutagenesis and subsequent selection. Two typical examples of these strategies are tRNA UTu [23] and tRNA SecUX [24]—engineered tRNAs that were developed for genetic encoding of selenocysteine.…”

Rational Design of Aptamer-Tagged tRNAs

“…Can the candidate tRNA chassis gain any amino-acid identity other than alanine and serine? To confirm the utility of the candidate tRNA chassis, the major identity elements for the archaeal-type tyrosyl-tRNA synthetase (TyrRS) (C1:G72 with A73) [ 43 , 44 , 45 ] were installed in the candidate tRNA chassis ( Figure 5 A), in order to develop synthetic Tyr tRNA molecules [ 44 ]. Y005, Y072, and Y073 variants were developed from S005, S072, and S073, respectively.…”