Structure elucidation by high resolution mass spectrometry of a highly modified nucleoside from mammalian transfer RNA. N-[(9-.beta.-D-Ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine

Yamaizumi, Ziro; Nishimura, Susumu; Limburg, K.; Raba, Manfred; Groß, H.; Crain, Pamela F.; McCloskey, James A.

doi:10.1021/ja00502a060

Cited by 28 publications

(26 citation statements)

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“…The identification of R as 2-methylthio-Nb-threonyladenosine has been achieved recently and will be reported elsewhere [63]. The other thionucleoside, designated as S and located in the first position of the anticodon, is mcm5s2U, which has also been found in a lysine tRNA from yeast [61].…”

Section: Discussionmentioning

confidence: 92%

“…Second, the major lysine tRNA species from SV40-transformed mouse fibroblasts cochromatograph precisely with the corresponding major mouse liver lysine tRNAs on reversed phase chromatography columns [63]; an as yet unidentified nucleoside K isolated from a B. suhtiliv lysine tRNA has an ultraviolet spectrum very similar to that of R from liver tRNAkY' and may therefore also be ms2t6A [70]. ( [57].…”

Section: Discussionmentioning

confidence: 99%

“…found in many eukaryotic tRNAs, this tRNA contains three other nucleosides of particular interest : Tm, which we have characterized before and shown to replace T in loop IV [59,60]; a nucleoside designated as R, which is located next to the anticodon and which we have characterized as 2-methylthio-N6-threonyladenosine (ms2t6A) [63] and a nucleoside designated as S, which we have characterized as 2-thio-5-carboxymethyluridine methyl ester (mcm5s2U) [61]. As will be seen below, S is the first nucleoside in the anticodon and was isolated as a dinucleotide from peak 4 of the pancreatic digest (Fig.…”

Section: Sequence Anulysis Of T R N A~~' F I O Mmentioning

confidence: 99%

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

Raba

Limburg

Burghagen

et al. 1979

European Journal of Biochemistry

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168

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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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Section: Discussionmentioning

confidence: 92%

Section: Discussionmentioning

confidence: 99%

Section: Sequence Anulysis Of T R N A~~' F I O Mmentioning

confidence: 99%

See 1 more Smart Citation

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

Raba

Limburg

Burghagen

et al. 1979

European Journal of Biochemistry

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168

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“…On the assumption that EDTA or other materials in an RPC-5 column eluate had suppressed the silylation reaction, the analysis was repeated using 5 pg (0.1 A260 unit) of tRNA that had been extracted from an acrylamide gel (B. Vold, unpublished work, 1980). As a result, ions characteristic of ribonucleosides, and in particular ms2t6A [26] (Fig.7), were observed. The diagnostic mass values used for identification were 774.2936 Da and 702.2539 Da, corresponding to M -C 2 H 4 0 from the pentasilyl (see structure) and tetrasilyl derivatives, respectively.…”

Section: Anulj~sis O F T R N a Hjdrolj~sutesmentioning

confidence: 93%

“…The reason for this is unclear, but may be due in part The present method was applied to the identification of the highly modified adenosine derivative ms2t6A [26] in tRNALyS from Bacillus suhtilis, in order to determine what limitations might result from samples isolated on relatively small scale by typical chromatographic procedures. Using total nucleoside digests difficulty had been previously encountered in identifying ms2t6A by conventional means [27], although its presence was suggested by the affinity of tRNAkY5 for an immunoadsorbent made with an antibody directed toward the related nucleoside t6A [27].…”

Section: Anulj~sis O F T R N a Hjdrolj~sutesmentioning

confidence: 99%

Identification of Nucleosides in Hydrolysates of Transfer RNA by High‐Resolution Mass Spectrometry

Pang

Smith

Crain

et al. 1982

European Journal of Biochemistry

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High‐resolution mass spectrometry has been investigated as a technique for identification of modified nucleosides in unfractionated hydrolysates of transfer RNA. The method is based on recognition of predetermined sets of exact mass values which are characteristic of individual nucleoside components. Mass spectra are photographically recorded in a non‐time‐resolved fashion, so that differing rates of nucleoside vaporization are of no consequence. Experimental parameters of sensitivity, spectrometer resolution and rates of vaporization were studied. Under typical conditions, 1–4 μg of tRNA are hydrolyzed, converted to volatile trimethylsilyl derivatives, and mass spectra of the resulting mixture recorded during 3 min at resolution 20000; 75–100% of the minor nucleosides are usually identified in a single recording. The method is complementary to conventional methods of identification which rely on chromatographic mobilities, and can in principle be generally applied to recognition of biologically or chemically modified bases in nucleic acid.

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Mass sectrometric study of methylene cross-linked derivatives of adenine and adenosine

Sethi

Gelijkens

McCloskey

et al. 1983

Biol. Mass Spectrom.

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Structure elucidation by high resolution mass spectrometry of a highly modified nucleoside from mammalian transfer RNA. N-[(9-.beta.-D-Ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine

Cited by 28 publications

References 1 publication

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

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

Identification of Nucleosides in Hydrolysates of Transfer RNA by High‐Resolution Mass Spectrometry

Mass sectrometric study of methylene cross-linked derivatives of adenine and adenosine

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