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Purification and properties of several transfer RNA methyltransferases fromS. typhimurium

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Summary

A fast method for a single-step fractionation of a number of tRNA methyltransferases fromSalmonella typhimurium is described. The method basically consists of ion-exchange chromatography on a phosphocellulose column and permits the separation of the enzymes forming mt6A, m1G, m5U, m7G. The enzyme fractions appear sufficiently purified to allow the estimation of some molecular and kinetic properties. The apparent KM for adenosylmethionine range between 1.5 to 3.2×10−5 M, whereas KM for undermethylated tRNA range between 3.1×10−5 M to 3.1×10−4 M. Glycerol gradient determination indicates the following Mr for the native proteins: 25×103, 40×103, 50×103 and 65×103 for m7G-, mt6A-, m1G- and m5U-forming enzymes, respectively. A complete analysis of methylated nucleosides formedin vivo inS. typhimurium has been obtained: it also allowed us to infer the pattern of the various tRNA methyltransferases for this prokaryote. The tRNA methyltransferase forming mt6A has been isolated for the first time from any type of cell.

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Abbreviations

m5U:

5-methyluridine

m7G:

7-methylguanosine

m1G:

1-methylguanosine

m2A:

2-methyladenosine

N6mA:

N6-methyladenosine

ms2i6A:

N6-(Δ2isopentenyl)-2-methylthioadenosine

mt6A:

N-[N′-methyl-N-(9-β-D-ribofuranosylpurin-6-yl)-carbomyl]-threonine

Um:

2′-O-methyluridine

Cm:

2′-O-methylcytidine

Gm:

2′-O-methylguanosine

N6mAde:

N6-methyladenine

m2Ade:

2-methyladenine

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Authors and Affiliations

  1. Istituto di Chimica Biologica, II Facoltà di Medicina e Chirurgia, Università di Napoli, Via Sergio Pansini 5, 80131, Napoli, Italy

    Filiberto Cimino, Cinzia Traboni, Alfredo Colonna, Paola Izzo & Francesco Salvatore

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  1. Filiberto Cimino
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  2. Cinzia Traboni
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  3. Alfredo Colonna
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  4. Paola Izzo
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  5. Francesco Salvatore
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Cimino, F., Traboni, C., Colonna, A. et al. Purification and properties of several transfer RNA methyltransferases fromS. typhimurium . Mol Cell Biochem 36, 95–104 (1981). https://doi.org/10.1007/BF02354908

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