Abstract
The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography. The polypeptide responsible for MTFmt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTFmt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTFmt consists of 357 amino acid residues. This sequence is about 30% identical to the corresponding Escherichia coli and yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTFmt formylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNAfMet) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNAmMet) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator Met-tRNAs. These observations are discussed in terms of the presence of a single tRNAMet gene in mammalian mitochondria.
Highlights
The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography
The E. coli elongator methionyl-tRNA (Met-tRNAmMet) was formylated with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E. coli methionyltRNA transformylase (MTF) which clearly discriminates between the bacterial initiator and elongator MettRNAs
The translational system in animal mitochondria is thought to be more closely related to that of prokaryotes than to that of the eukaryotic cell cytoplasm [7, 8]. This idea is based on the use of fMet-tRNA for initiation, on the antibiotic sensitivity of the ribosomes, and on the ability of the mammalian mitochondrial elongation factors to function on bacterial ribosomes
Summary
Folinic acid and CHAPS were purchased from Sigma. [35S]Methionine (37 TBq/mmol) and [14C]methionine (1.85 GBq/mmol) were obtained from Amersham. Folinic acid and CHAPS were purchased from Sigma. [35S]Methionine (37 TBq/mmol) and [14C]methionine (1.85 GBq/mmol) were obtained from Amersham. DEAE-Sepharose fast flow, Mono S (HR5/5), Hi Trap Blue, and Hi Trap Heparin columns were purchased from Pharmacia. An affinity column using an immobilized E. coli tRNA mixture was prepared as described [14]. Buffer TG contains 20 mM Tris-HCl (pH 7.6), 1 mM MgCl2, 0.1 mM EDTA, 6 mM -mercaptoethanol, 10% glycerol, 0.1 mM phenymethylsulfonyl fluoride. Buffer PG contains 20 mM potassium phosphate (pH 6.8), 10% glycerol, 1 mM dithiothreitol, 0.1 mM phenymethylsulfonyl fluoride, 0.5% CHAPS
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