Purification and Characterization of tRNA (Adenine‐1‐)‐Methyltransferase from Thermus flavus Strain 71

Abstract

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].

In silico analysis of the tRNA:m1A58 methyltransferase family: homology‐based fold prediction and identification of new members from Eubacteria and Archaea

“…I predict that the two‐subunit yeast tRNA:m 1 A58 MTase evolved from a duplicated, possibly dimeric enzyme, in which one of the subunits lost the ability to bind tRNA on its own and in the other the catalytic site degenerated. The hypothesis of the dimeric structure of prokaryotic enzymes is in good agreement with the experimental estimation of the MW for tRNA:m 1 A58 MTases from Thermus flavus [30] and Thermus thermophilus [31] (78 kDa and 60 kDa, respectively). No sequences of Gcd14p homologs are available from these organisms, however the calculated MW for all sequences of predicted prokaryotic tRNA:m 1 A58 MTase analyzed in this work are in the range of 27.8 kDa ( Aeropyrum pernix ) to 35.2 kDa ( Rhodococcus erythropolis ).…”

The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

“…and A.G.H., unpublished observations), suggesting that Gcd10p-and Gcd14p-related proteins comprise the tRNA(m 1 A)MTase in diverse eukaryotes. Purification of tRNA(m 1 A)MTases from bovine liver (19) and extreme thermophilic archaebacteria (20,21) indicated that the activity resided within single polypeptides of 95 kDa and 78 or 60 kDa, respectively; however, the genes encoding these proteins have not been described. It is possible that the bovine enzyme represents a higher molecular weight Gcd14p homolog that does not require a Gcd10p-related subunit for activity.…”

RNA recognition mechanism of eukaryote tRNA (m7G46) methyltransferase (Trm8–Trm82 complex)

“…This enzyme activity was first detected in a cell extract of Escherichia coli [8] and has been purified more than 1000 fold [9]. The enzyme activity has also been purified from Salmonella typhimurium [10] and Thermus flavus [11]. Furthermore, the tRNA m 7 G46 modification activity has been detected in crude extracts from higher eukaryotes [12–14].…”