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.
Highlights
An L-shape form is the prominent structural feature of tRNA and is maintained through tertiary interactions taking place in the tRNA core, which encompasses the D arm, the T loop, and the intervening nucleotides between the four arms [1,2] (Figure 1)
The D and T loops are bound together by the invariant G18-ψ55 and G19-C56 base pairs (“ψ” is pseudouridine, a modified U), while the D stem and its surrounding nucleotides are integrated into a structure called the augmented D helix [2]
When Mj tRNATyr was converted into an amber suppressor (MJR, Table 1), the resulting tRNA reportedly showed a low level of suppressor activity in the absence of Mj TyrRS in E. coli [16]
Summary
An L-shape form is the prominent structural feature of tRNA and is maintained through tertiary interactions taking place in the tRNA core, which encompasses the D arm, the T loop, and the intervening nucleotides between the four arms [1,2] (Figure 1). An important effect of the tRNA folding into the L shape is to place the anticodon and 3’-terminal CCA sequence at the opposite extremities, with the correct distance and relative orientations between them. When Mj tRNATyr was converted into an amber suppressor (MJR, Table 1), the resulting tRNA reportedly showed a low level of suppressor activity in the absence of Mj TyrRS in E. coli [16]. PYLYCMa showed a higher suppression activity than PYLY1 and PYLY2, while PYLYDh was aminoacylated by Mj TyrRS, its suppressor activity was lower than that of PYLY1 (Table 1 and Figure S1B) These observations indicated that the tRNAPyl scaffold recognized by the archaeal TyrRS is not limited to the particular sequence of Mm tRNAPyl
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