Mitochondrial methionyl-tRNAfMet formyltransferase from Saccharomyces cerevisiae: gene disruption and tRNA substrate specificity.
Initiation of protein synthesis in bacteria, mitochondria, and chloroplasts involves a formylated methionyl-tRNA species. Formylation of this tRNA is catalyzed by a methionyl-tRNA(f)(Met) formyltransferase (formylase). Upon inactivation of the gene encoding formylase, the growth rate of Escherichia coli is severely decreased. This behavior underlines the importance of formylation to give tRNA(Met) an initiator identity. Surprisingly, however, recent data [Li, Y., Holmes, W. B., Appling, D. R., and RajBhandary, U. L. (2000) J. Bacteriol. 182, 2886-2892] showed that the respiratory growth of Saccharomyces cerevisiaewas not sensitive to deprivation of the mitochondrial formylase. In the present study, we report conditions of temperature or of growth medium composition in which inactivation of the formylase gene indeed impairs the growth of a S. cerevisiae haploid strain. Therefore, some selective advantage can eventually be associated to the existence of a formylating activity in the fungal mitochondrion under severe growth conditions. Finally, the specificity toward tRNA of S. cerevisiae mitochondrial formylase was studied using E. coli initiator tRNA and mutants derived from it. Like its bacterial counterpart, this formylase recognizes nucleotidic features in the acceptor stem of mitochondrial initiator tRNA. This behavior markedly distinguishes the mitochondrial formylase of yeast from that of animals. Indeed, it was shown that bovine mitochondrial formylase mainly recognizes the side chain of the esterified methionine plus a purine-pyrimidine base pair in the D-stem of tRNA [Takeuchi, N., Vial, L., Panvert, M., Schmitt, E., Watanabe, K., Mechulam, Y., and Blanquet, S. (2001) J. Biol. Chem. 276, 20064-20068]. Distinct tRNA recognition mechanisms adopted by the formylases of prokaryotic, fungal, or mammalian origins are likely to reflect coevolution of these enzymes with their tRNA substrate. Each mechanism appears well suited to an efficient selection of the substrate within the pool of all tRNAs.
Vial
Lionel
L
Laboratoire de Biochimie, Unité Mixte de Recherche 7654, CNRS-Ecole Polytechnique, F-91128 Palaiseau Cedex, France.
Gomez
Pilar
P
Panvert
Michel
M
Schmitt
Emmanuelle
E
Blanquet
Sylvain
S
Mechulam
Yves
Y
D000595:Amino Acid Sequence; D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D002384:Catalysis; D002417:Cattle; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D004952:Esters; D020868:Gene Silencing; D005800:Genes, Fungal; D005822:Genetic Vectors; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry* / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010101:Oxygen Consumption / Q000235:genetics; D012358:RNA, Transfer, Met / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics*; D013379:Substrate Specificity / Q000235:genetics
D029968:Escherichia coli Proteins; D004952:Esters; D012358:RNA, Transfer, Met; D029701:Saccharomyces cerevisiae Proteins; D019877:Hydroxymethyl and Formyl Transferases; C021561:methionyl-tRNA formyltransferase