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. 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.

Relative utilization of mammalian Lys‐tRNA isoacceptors in protein synthesis

“…The large peak which eluted first from the column (designated Peak I) recognizes AAG [4-61. This peak contains two isoacceptors which are not separated by RPC-5 chromatography [4]. The large, second eluting peak (designated Peak II) is Lys-tRN& and, like Peak I, it recognizes AAG [4-61.…”

“…Raba et al [4] recently sequenced this and other mammalian lysine isoacceptors and demonstrated that Lys-tRNA4 is an underm~i~~d form of one of the other Lys-tRNAs. Although Ortwerth et al [S] showed that lysine can be incorporated into protein from Lys-tRNA4, neither they nor Raba et al [4] compared the utilization of this isoacceptor in protein synthesis relative to other lysine isoacceptors which have the same codon recognition properties 14-63. In the present study, the relative utilization of these lysine isoacceptors during globin synthesis in a wheat-germ extract was determined.…”

tRNALys3: The Primer tRNA for Reverse Transcription in HIV‐1

“…tRNA Trp is the primer for avian retroviruses, and tRNA Pro is a common primer for murine retroviruses. There are three major tRNA Lys isoacceptors in mammalian cells (2). tRNA Lys1,2 , representing two tRNA Lys isoacceptors differing by one base pair in the anticodon stem, is also the primer tRNA for several retroviruses, including Mason-Pfizer monkey virus (MPMV) and human foamy virus (HFV).…”

The nucleotide sequence of lysine tRNA2from Drosophila

“…It is pertinent to note that it is exactly the same sequence as found for the same tRNA isoacceptor in rabbit liver and presumably other mammalian organisms (19).…”