Mammalian Mitochondrial Methionyl-tRNA Transformylase from Bovine Liver

Abstract

The mammalian mitochondrial methionyl-tRNA transformylase (MTF mt ) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography. The polypeptide responsible for MTF mt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTF mt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTF mt 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 MTF mt formylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNA fMet ) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNA mMet ) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTF mt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator MettRNAs. These observations are discussed in terms of the presence of a single tRNA Met gene in mammalian mitochondria.

Eukaryotic-type elongator tRNA Met of Trypanosoma brucei becomes formylated after import into mitochondria

“…Thus, the substrate specificity of the trypanosomal MTF is diametrically opposed to bacterial or mitochondrial MTFs, which in vivo only formylate initiator and not elongator tRNAs Met . The bovine mitochondrial MTF, which is the only mitochondrial MTF characterized so far (21), has a relaxed substrate specificity when compared with the bacterial enzyme, especially concerning the acceptor stem region of the substrate tRNA Met (3). The trypanosomal elongator tRNA Met-e , however, is devoid of all of the known structural features that are recognized by the mitochondrial MTF in the bovine system.…”

Mammalian Mitochondrial Initiation Factor 2 Supports Yeast Mitochondrial Translation without Formylated Initiator tRNA

“…In eubacteria, the initiator Met-tRNA f Met is formylated by the enzyme methionyl-tRNA formyltransferase. Formylated methionyl-tRNA (fMet-tRNA f Met ), along with MettRNA formyltransferase activity, is also observed in mitochondria of eukaryotes such as Saccharomyces cerevisiae, Neurospora crassa, and mammals (2)(3)(4)(5). These observations, along with the identification of formylmethionine at the N terminus of several mitochondrially synthesized proteins (6), led to the widely accepted dogma that mitochondrial protein synthesis initiation requires fMet-tRNA f Met in a process involving IF2.…”

Search for characteristic structural features of mammalian mitochondrial tRNAs

“…The outstanding level of primary sequence conservation within the methionine tRNAs, and as a consequence, the high conservation in secondary and tertiary structures of these tRNAs, make the tRNA Met family a unique case+ Interestingly, there is only one type of tRNA Met encoded in the mammalian mt DNAs, so that a single molecule has to serve both as initiator and elongator tRNA+ Thus, apart from being methionylated by methionyl-tRNA synthetase, this single tRNA must on one hand, be recognized by translation initiation factors and the mt methionyl-tRNA transformylase, and be able to read start codons of the mt mRNAs, and, on the other hand, the unformylated methionyl-tRNA must be specifically recognized by the elongation complex and be able to decode elongation codons+ This need for a relatively high number of different interactions undergone by mt tRNA Met , as compared to the other mt tRNAs, is likely the reason for their high degree of conservation+ Each interaction with a specific protein would require different conserved features+ The two conserved T50-T64 and Y51-N63 mismatches in the T-stem and a A5-R68 mismatch in the acceptor stem are features that have so far not been identified in any other tRNA, and are therefore prime candidates as signals for recognition (or against recognition) by either of the above mentioned proteins+ Among the different deviations from the universal genetic code found in mitochondria (Watanabe & Osawa, 1995) is the AUA codon, otherwise assigned to isoleucine, which has been taken over by methionine+ A unique posttranscriptional modification of C34 has been identified as 5-formylcytidine and is thought to be responsible for the unique decoding properties (Moriya et al+, 1994;Takemoto et al+, 1995)+ Because the mammalian initiation and elongation factors (Ma & Spremulli, 1995;Zhang & Spremulli, 1998) and the mt methionyl-tRNA transformylase (Takeuchi et al+, 1998) are known, the MetRS seems to be the only missing piece to solve these highly interesting mechanisms+…”