Production of yeast tRNA (m(7)G46) methyltransferase (Trm8-Trm82 complex) in a wheat germ cell-free translation system.
Cell-free translation systems are a powerful tool for the production of many kinds of proteins. However the production of proteins made up of hetero subunits is a major problem. In this study, we selected yeast tRNA (m(7)G46) methyltransferase (Trm8-Trm82 heterodimer) as a model protein. The enzyme catalyzes a methyl-transfer from S-adenosyl-l-methionine to the N(7) atom of guanine at position 46 in tRNA. When Trm8 or Trm82 mRNA were used for cell-free translation, Trm8 and Trm82 proteins could be synthesized. Upon mixing the synthesized Trm8 and Trm82 proteins, no active Trm8-Trm82 heterodimer was produced. Active Trm8-Trm82 heterodimer was only synthesized under conditions, in which both Trm8 and Trm82 mRNAs were co-translated. These results strongly suggest that the association of the Trm8 and Trm82 subunits is translationally controlled in living cells. Kinetic parameters of purified Trm8-Trm82 heterodimer were measured and these showed that the protein has comparable activity to other tRNA methyltransferases. The production of the m(7)G base at position 46 in tRNA was confirmed by two-dimensional thin layer chromatography and aniline cleavage of the methylated tRNA.
Matsumoto
Keisuke
K
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan.
Tomikawa
Chie
C
Toyooka
Takashi
T
Ochi
Anna
A
Takano
Yoshitaka
Y
Takayanagi
Naoyuki
N
Abe
Masato
M
Endo
Yaeta
Y
Hori
Hiroyuki
H
D001483:Base Sequence; D002474:Cell-Free System; D019281:Dimerization; D008954:Models, Biological; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014908:Triticum / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D012343:RNA, Transfer; D012359:tRNA Methyltransferases; C036814:tRNA (guanine-N7-)-methyltransferase