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 m7G methyltransferase are temperature-sensitive in synthetic media containing glycerol. Bacterial TRM8 orthologs complement the growth defect of trm8-Delta, trm82-Delta, and trm8-Delta 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 m7G methyltransferase activity in vitro and in vivo, based on analysis of 10 mutant alleles of trm8 and bacterial orthologs, and suggests that m7G 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 m7G modification, and that a major role of Trm82p is to maintain cellular levels of Trm8p. Trm8p efficiently cross-links to pre-tRNAPhe in vitro in the presence or absence of Trm82p, in addition to its known residual tRNA m7G 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.
Alexandrov
Andrei
A
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, 601 Elmwood Ave., Box 712, Rochester, NY 14642, USA.
Grayhack
Elizabeth J
EJ
Phizicky
Eric M
EM
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D002384:Catalysis; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D005816:Genetic Complementation Test; D005990:Glycerol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D012322:RNA Precursors / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development*; D013696:Temperature; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003470:Culture Media; D012322:RNA Precursors; D012360:RNA, Transfer, Phe; D016601:RNA-Binding Proteins; D012359:tRNA Methyltransferases; C036814:tRNA (guanine-N7-)-methyltransferase; D005990:Glycerol