Abstract
Reprogramming of the genetic code system is limited by the difficulty in creating new tRNA structures. Here, I developed translationally active tRNA variants tagged with a small hairpin RNA aptamer, using Escherichia coli reporter assay systems. As the tRNA chassis for engineering, I employed amber suppressor variants of allo-tRNAs having the 9/3 composition of the 12-base pair amino-acid acceptor branch as well as a long variable arm (V-arm). Although their V-arm is a strong binding site for seryl-tRNA synthetase (SerRS), insertion of a bulge nucleotide in the V-arm stem region prevented allo-tRNA molecules from being charged by SerRS with serine. The SerRS-rejecting allo-tRNA chassis were engineered to have another amino-acid identity of either alanine, tyrosine, or histidine. The tip of the V-arms was replaced with diverse hairpin RNA aptamers, which were recognized by their cognate proteins expressed in E. coli. A high-affinity interaction led to the sequestration of allo-tRNA molecules, while a moderate-affinity aptamer moiety recruited histidyl-tRNA synthetase variants fused with the cognate protein domain. The new design principle for tRNA-aptamer fusions will enhance radical and dynamic manipulation of the genetic code.
Highlights
RNA aptamers are small RNA molecules that bind to their target molecules
In an attempt to develop a seryl-transfer RNAs (tRNAs) synthetase (SerRS)-rejecting (9/3) allo-tRNASer variant, we realized that certain allo-tRNA species having a G:U wobble base pair at the bottom of the V-stem worked as less active tRNASer than allo-tRNA variants carrying Watson-Crick (WC) changes
In the presence of Ma TyrRS, Y005M translated the amber codon of the sfGFP(Tyr66TAG) reporter gene with tyrosine (Figure 5F). These results clearly demonstrated that SerRS-rejecting allo-tRNA chassis can be engineered and converted to synthetic Tyr tRNA species
Summary
RNA aptamers are small RNA molecules that bind to their target molecules. The V-arm hairpin of a yeast serine tRNA species was replaced by the MS2 phage hairpin RNA (19 nucleotides in length) to recruit an enzyme fused with the MS2 coat protein on the premature form of these fusion RNA molecules in yeast cells [11]. These works encouraged us to design a new generation of tRNA-aptamer fusions in which a small hairpin aptamer constitutes the tip of the V-arm
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