Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus

Hironori Okamoto, Kazunori Watanabe, Yoshiho Ikeuchi, Tsutomu Suzuki, Yaeta Endo, Hiroyuki Hori

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Transfer RNA (m7G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m7G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNA Phe and E. coli tRNAThr. It was confirmed that 5′-leader and 3′-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.

Original languageEnglish
Pages (from-to)49151-49159
Number of pages9
JournalJournal of Biological Chemistry
Volume279
Issue number47
DOIs
Publication statusPublished - Nov 19 2004
Externally publishedYes

Fingerprint

tRNA Methyltransferases
Methyltransferases
Transfer RNA
RNA Precursors
Escherichia coli
RNA, Transfer, Thr
Substrates
RNA, Transfer, Phe
S-Adenosylmethionine
Proteins
Guanine
RNA
Enzymes
Nucleosides
Liquid Chromatography
Open Reading Frames
Gene encoding
Light trailers
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence

ASJC Scopus subject areas

  • Biochemistry

Cite this

Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus. / Okamoto, Hironori; Watanabe, Kazunori; Ikeuchi, Yoshiho; Suzuki, Tsutomu; Endo, Yaeta; Hori, Hiroyuki.

In: Journal of Biological Chemistry, Vol. 279, No. 47, 19.11.2004, p. 49151-49159.

Research output: Contribution to journalArticle

Okamoto, Hironori ; Watanabe, Kazunori ; Ikeuchi, Yoshiho ; Suzuki, Tsutomu ; Endo, Yaeta ; Hori, Hiroyuki. / Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus. In: Journal of Biological Chemistry. 2004 ; Vol. 279, No. 47. pp. 49151-49159.
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