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 tRNA^Pyl. First, we constructed a naïve hybrid between archaeal tRNA^Pyl and tRNA^Tyr, which consisted of the acceptor and T stems of tRNA^Tyr and the other parts of tRNA^Pyl. 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 tRNA^Tyr suppressor assuming the canonical structure. After a series of modifications to this hybrid tRNA, we obtained two artificial types of tRNA^Tyr: 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.

Keywords: amber suppression; genetic code expansion; pyrrolysine tRNA; tRNA secondary structure; tertiary base pairs.

Figure 1

The cloverleaf (A) and L-shape (B) representations of the wild-type M. jannaschii tRNA^Tyr, which belongs to class I tRNA. The nucleotides are all indicated in unmodified forms, and the invariant T54 and ψ55 are shown as U. The invariant nucleotides mentioned in the text are indicated in gray boxes, and the tertiary base pairs between them are represented by broken lines (A) or the extension of the gray boxes (B). Nucleotides are numbered by the standard system.

Figure 2

L-shape representations of M. mazei (Mm) tRNA^Pyl, PYLY1 (the naïve hybrid between Mm tRNA^Pyl and M. jannaschii tRNA^Tyr), and two representative variants isolated from selections. The nucleotides in blue and black are derived from M. jannaschii tRNA^Tyr and Mm tRNA^Pyl, respectively. The nucleotides in red show randomized positions. The identity elements of tRNA^Pyl and tRNA^Tyr are underlined. A tertiary base pair in Mm tRNA^Pyl and presumed tertiary base pairs in the other tRNAs are indicated with grey boxes. Some nucleotides are numbered to highlight the difference between our numbering system and the standard one.

Figure 3

In vivo amber suppression activities of MJR1 and PYLY1. The cell suspensions of two E. coli clones transformed with each of the indicated variant genes were diluted successively (from top to bottom) and grown on the rich medium plates containing Cm at the indicated final concentrations with or without the supplementation of 3-iodotyrosine (IY).