Nucleotide Sequence of Three Isoaccepting Lysine tRNAs from Rabbit Liver and SV40‐Transformed Mouse Fibroblasts

Abstract

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a ‘new’ lysine tRNA, which is practically absent in non-dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the ‘new’ lysine tRNA isolated from SV40-transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows. a) The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2′-O-methyl-ribosylthymine (Tm) in place of T. tRNA₁^Lys and tRNA₂^Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C-U-U is followed by N-threonyl-adenosine (t⁶A). tRNA₃^Lys has the anticodon S-U-U and contains two highly modified thionucleosides, S (shown to be 2-thio-5-carboxymethyl-uridine methyl ester) and a further modified derivative of t⁶A (2-methyl-thio-MN⁶-threonyl-adenosine) on the 3′ side of the anticodon. tRNA₃^Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors. b) Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRN₂^Lys with anticodon C-U-U recognized A-A-G only, whereas tRNA₃^Lys, which contains thionucleotides in and next to the anticodon, decodes both lysine codons A-A-G and A-A-A, but with a preference for A-A-A. In a globin-mRNA-translating cell-free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA₂^Lys than with tRNA₃^Lys, in agreement with the fact that α-globin and β-globin mRNAs contain more A-A-G than A-A-A codons for lysine. c) A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the ‘new’ tRNA₄^Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA₂^Lys. Of the two dihydrouridines present in tRNA₂^Lys, one is found as U in tRNA₄^Lys; the purine next to the anticodon is as yet unidentified but is known not to be t⁶A. In addition we have found U, T and Ψ besides Tm as the first nucleoside in loop IV.