Identification and Conversion of Molecular Recognition Elements in Yeast Tyrosine tRNA

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(1) When yeast tyrosine tRNA was used in a cell-free protein-synthesizing system derived from E. coli, it was shown to be charged slightly with lysine by E. coli lysyl-tRNA synthetase (LysRS). Therefore, the primary target of this project was set to find out a way to protect the tRNA from mis-acylation by E. coli LysRS.(2) Several variants of yeast tyrosine tRNA, which have potentially negative identity elements against E. coli LysRS, were synthesized by our new method using transcription with T7 RNA polymerase and processing with RNase P. Aminoacylation activities of these tRNA variants were measured with yeast tyrosyl-tRNA synthetase (TyrRS) for normal tyrosine acceptance and E. coli LysRS for mis-acylation with lysine.(3) A variant tRNA, in which 3 A-U base pairs in the anticodon stem were replaced with 3 G-C base pairs, was shown to be drastically reduced in the mis-acylation, while keeping the original tyrosine-accepting ability. Thus these variations are proved to be actually part of the negative identity elements against E. coli LysRS.(4) Conversion of the amino acid specificity of yeast TyrRS was attempted by using genetic engineering and a substantial progress was achieved.(5) An unnatural (non-Watson-Crick type) base pair was developed by inserting one base (2-amino-6-(2-thienyl)purine) in the anticodon of yeast tyrosine tRNA and the other one (pyridin-2-one) in the transcribed ras mRNA.