tRNA m7G methyltransferase Trm8p/Trm82p:: Evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p

We show that Saccharomyces cerevisiae strains lacking Trm8p/Trm82p tRNA m(7)G methyltransferase are temperature-sensitive in synthetic media containing glycerol. Bacterial TRM8 orthologs complement the growth defect of trm8-Delta, trm82-Delta, and trm8-A trm82-Delta double mutants, suggesting that bacteria employ a single subunit for Trm8p/Trm82p function. The growth phenotype of trm8 mutants correlates with lack of tRNA m(7)G methyltransferase activity in vitro and in vivo, based on analysis of 10 mutant alleles of trmB and bacterial orthologs, and suggests that m(7)G modification is the cellular function important for growth. Initial examination of the roles of the yeast subunits shows that Trm8p has most of the functions required to effect m(7)G modification, and that a major role of Trm82p is to maintain cellular levels of Trm8p. Trm8p efficiently cross-links to pre-tRNA(Phe) in vitro in the presence or absence of Trm82p, in addition to its known residual tRNA m(7)G modification activity and its SAM-binding domain. Surprisingly, the levels of Trm8p, but not its mRNA, are severely reduced in a trm82-Delta strain. Although Trm8p can be produced in the absence of Trm82p by deliberate overproduction, the resulting protein is inactive, suggesting that a second role of Trm82p is to stabilize Trm8p in an active conformation.