The substrate specificity of tRNA (m1G37) methyltransferase (TrmD) from Aquifex aeolicus.
Transfer RNA (m(1)G37) methyltransferase (TrmD) catalyzes methyl-transfer from S-adenosyl-L-methionine to the N(1) atom of G37 in tRNA. In Escherichia coli cells, TrmD methylates tRNA species possessing a G36G37 sequence. It was previously believed that G36 was the positive determinant of TrmD recognition. In the current study, we demonstrate that TrmD from Aquifex aeolicus methylates tRNA transcripts possessing an A36G37 sequence as well as tRNA transcripts possessing a G36G37 sequence. In contrast, tRNA transcripts possessing pyrimidine36G37 were not methylated at all. These substrate specificities were confirmed by an in vitro kinetic assay using 16 tRNA transcripts. The modified nucleoside and the position in yeast tRNA(Phe) transcript were confirmed by LC/MS. Furthermore, nine truncated tRNA molecules were tested to clarify the additional recognition site. Unexpectedly, A. aeolicus TrmD protein efficiently methylated the micro helix corresponding to the anti-codon arm. Because the disruption of the anti-codon stem caused the complete loss of the methyl group acceptance activity, the anti-codon stem is essential for the recognition. Moreover, the existence of the D-arm structure inhibited the activity. Recently, it was reported that E. coli TrmD methylates yeast tRNA(Phe) harboring a sequence A36G37. Thus, recognition of the purine36G37 sequence is probably common to eubacteria TrmD proteins.
Takeda
Hiroshi
H
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan.
Toyooka
Takashi
T
Ikeuchi
Yoshiho
Y
Yokobori
Shin-ichi
S
Okadome
Kan
K
Takano
Fuyumi
F
Oshima
Tairo
T
Suzuki
Tsutomu
T
Endo
Yaeta
Y
Hori
Hiroyuki
H
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D013058:Mass Spectrometry; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012359:tRNA Methyltransferases