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. The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2′‐O‐methyl‐ribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys 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). tRNA3Lys 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. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors. Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRN2Lys with anticodon C‐U‐U recognized A‐A‐G only, whereas tRNA3Lys, 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 tRNA2Lys than with tRNA3Lys, in agreement with the fact that α‐globin and β‐globin mRNAs contain more A‐A‐G than A‐A‐A codons for lysine. A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the ‘new’ tRNA4Lys 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 tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; 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.

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One of the lysine transfer RNAs of rabbit liver is shown to contain 2'-O-methyl ribothymidine in place of ribothymidine. This represents the first demonstration of the presence of 2'-O-methyl ribothymidine in a nucleic acid.

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