Production of yeast (m2G10) methyltransferase (Trm11 and Trm112 complex) in a wheat germ cell-free translation system.

Transfer RNA (guanine-N(2)-)-methyltransferase [tRNA (m(2)G10) methyltransferase] catalyzes a methyl-transfer from S-adenosyl-L-methionine to N(2)-atom of guanine at position 10 (G10) in tRNA and generates N(2)-methylguanine at position 10 (m(2)G10). Yeast enzyme contains two protein subunits (Trm11 and Trm112). Trm11 protein is expected to be a catalytic subunit and Trm112 contains a Zinc-finger. In yeast cells, Trm112 binds not only to Trm11 but also to other proteins such as Lys9, Trm9, and Mtq2. Therefore, the Trm112 protein may regulate population of several protein complexes. To address these issues, we started the study on synthesis of Trm112 related protein complexes. In this meeting, we report synthesis of active Trm11-Trm112 complex in a wheat germ cell-free translation system.

Hetero subunit interaction and RNA recognition of yeast tRNA (m7G46) methyltransferase synthesized in a wheat germ cell-free translation system.

Yeast tRNA (m(7)G46) methyltransferase contains two protein subunits (Trm8 and Trm82). The enzyme catalyzes a methyl-transfer from S-adenosyl-L-methionine to the N(7) atom of guanine at position 46 in tRNA. We deviced synthesis of active Trm8-Trm82 heterodimer in a wheat germ cell-free translation system. When Trm8 or Trm82 mRNA were used for a synthesis, Trm8 or Trm82 protein 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. To address the RNA recognition mechanism of the Trm8-Trm82 complex, we investigated methyl acceptance activities of eight truncated yeast tRNA(Phe) transcripts. In this meeting, we demonstrate that yeast Trm8-Trm82 has stricter recognition requirements for the tRNA molecule as compared to the bacterial enzyme, TrmB.