tRNA (adenine-I-)-methyltransferase was isolated from the extreme thermophile Thermus ~~X L Y , strain 71. It was purified about 2000-fold by ammonium sulhte fractionation and affinity chromatography on tRNA bound to aminohydroxybutylcellulose via its oxidized 3' end. The purified protein preparation is free of nuclease and aminoacyl-tRNA synthetase activity and contains no more than 4 of tRNA (guanine-7-)-methyltransferase activity. The only activity of the enzyme is to methylate As* in the T Y loop of tRNA. Out of the eight purified tRNAs examined, only yeast tRNATrp was not utilized as a substrate. The enzyme is highly thermostable. It is most active at 75 "C. tRNA (adenine-I-)-methyltransferase has a K , of 0.4-0.5 pM for tRNA9'" from Escherichia coli and a K , of 6 pM for S-adenosyl-L-methionine.A characteristic feature of tRNAs is a high content of modified nucleotides in particular methylated ones. Methyl groups are transferred to tRNAs by tRNA methyltransferases and AdoMet usually serves as a donor of the methyl group. Although tRNA methyltransferases were discovered as far back as 1962, progress towards understanding their function and interaction with tRNA has been very slow, due to difficulties in their isolation in the purified state. tRNA methylases compose a group of highly specific enzymes; the separation of a particular methylase from all others is a difficult task [2]. Even in tissues in which methylases are most active, the content of each of them is very low. Finally methylases are highly labile proteins and each purification step leads to a more or less significant inactivation. Several laboratories have succeeded purifying methylases [3 -71. We need purified methylases for studying their interaction with tRNA. As a source, we chose the extreme thermophile Thermus flavus strain 71 for the following reasons. First, one might expect a higher stability of protein from this organism which exists at 80 "C. Second, the number of methylases in T . flavus, a prokaryote, is low in comparison with eukaryotes [8].We describe in this article the purification of tRNA (adenine-I-)-methyltransferase from T . ,flavus strain 71 and some of its properties.Ahhrrvicr/ions. AdoMet, S-adenosyl-L-methionine; A d~[ M r -'~c ] -Met, S-adenosyI-~-[mrthyl-'~C]methionine; mlA, 1-methyladenosine; m7G, 7-methylguanosine; Gm, 2'-O-methylguanosine; s2T, 2-thioribosylthymine: D, 5,h-dihydrouridine; m'A methylase, tRNA (adenine-1-)-methyl transferase; m7G methylase, tRNA (guanine-7-)-methyltransferase; ABC, aminohydroxybutylcellulose; ABC -oxidized-tRNA, ABC coupled with oxidized tRNA; ABC-rRNA, ABC coupled with tRNA; SDS, sodium dodecyl sulphate; PEI-cellulose poly(ethy1eneimine)-cellulose. The numbering of nucleotides in tRNAs is that of cytoplasmic yeast tRNAPhe [l].
