title: Substrate specificity of tRNA (adenine-1-)-methyltransferase from Thermus thermophilus HB27.
abstract: tRNA (adenine-1-)-methyltransferase was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB27, by several steps of column chromatographies. The molecular weight of this enzyme was about 60,000 as analyzed by SDS polyacrylamide gel electrophoresis. Km for E. coli tRNA(2Glu) was 100 nM and that for the methyl group donor, S-adenosyl-L-methionine, was 7.8 microM. The substrate specificity of the enzyme was investigated by using T7 RNA polymerase transcripts and tRNA fragments obtained by partial digestion with RNases. The enzyme was able to transfer the methyl group to the 3'-half fragment of E. coli initiator tRNA, however, the extent of methylation was elevated by more than five times when the 5'-half fragment was added and annealed to the 3'-half. This indicates that the main recognition site of the enzyme is within the 3'-half region of tRNA molecule, while the tertiary interaction between the T-loop and the D-loop is very effective for the adequate methylation reaction.
authors.affiliation: Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Yamazaki
authors.forename: H
authors.initials: H
authors.lastname: Hori
authors.forename: K
authors.initials: K
authors.lastname: Ozawa
authors.forename: S
authors.initials: S
authors.lastname: Nakanishi
authors.forename: T
authors.initials: T
authors.lastname: Ueda
authors.forename: I
authors.initials: I
authors.lastname: Kumagai
authors.forename: K
authors.initials: K
authors.lastname: Watanabe
authors.forename: K
authors.initials: K
authors.lastname: Nishikawa
mesh_terms: D001483:Base Sequence; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000201:enzymology*; D012359:tRNA Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D012343:RNA, Transfer; D012359:tRNA Methyltransferases; C023194:tRNA (adenine(58)-N(1))-methyltransferase