Hydroxyl radical probing of tRNA (Gm18) methyltransferase [TrmH]-AdoMet-artificial tRNA ternary complex.
Transfer RNA (Gm18) methyltransferase [TrmH] catalyzes methyl-transfer from S-adenosyl-L-methionine (AdoMet) to the 2'-OH of ribose of the conserved G18 in tRNA. In a previous study, we demonstrated that the affinity of the enzyme for tRNA is enhanced in the presence of AdoMet and that the C-terminal region (alpha8-helix) is important for tRNA binding. In this symposium we report the successful preparation of the TrmH-AdoMet-tRNA ternary complex using artificial tRNA molecules, which contain deoxyribonulcotide(s). We used the ternary complex to identify the interaction sites between the C-terminal region and tRNA. In order to be able to employ Fe(III) (s)-1-(p-bromoacetamidobenzyl) ethlenediaminetetraacetic acid (Fe-BABE) as a chemical modification probe, we prepared three double mutant proteins, which were substituted at Asp180, Asp190, or Trp191 by cysteine and at Cys43 by alanine. We tried to identify the creavage sites of the ternary complex of Fe-BABE modified mutant protein-AdoMet-artificial tRNA induced by ascorbic acid. In this meeting, we demonstrate the interaction between the C-terminal region of the TrmH and artificial tRNA.
Ochi
Anna
A
Department of Materials Science and Biotechnology, Graduate School of Science and Engineering Ehime University, Bunkyo 3, Matuyama 790-8577, Japan.
Hori
Hiroyuki
H
D001665:Binding Sites; D003848:Deoxyguanine Nucleotides / Q000737:chemistry; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D017665:Hydroxyl Radical / Q000737:chemistry; D007700:Kinetics; D009154:Mutation; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D003848:Deoxyguanine Nucleotides; D017665:Hydroxyl Radical; D012436:S-Adenosylmethionine; C042007:1-(4-bromoacetamidobenzyl)EDTA; D012343:RNA, Transfer; D004492:Edetic Acid; D012359:tRNA Methyltransferases