Yeast AEP3p is an accessory factor in initiation of mitochondrial translation.

Initiation of protein synthesis in mitochondria and chloroplasts normally uses a formylated initiator methionyl-tRNA (fMet-tRNA(f)(Met)). However, mitochondrial protein synthesis in Saccharomyces cerevisiae can initiate with nonformylated Met-tRNA(f)(Met), as demonstrated in yeast mutants in which the nuclear gene encoding mitochondrial methionyl-tRNA formyltransferase (FMT1) has been deleted. The role of formylation of the initiator tRNA is not known, but in vitro formylation increases binding of Met-tRNA(f)(Met) to translation initiation factor 2 (IF2).

Identification and characterization of archaeal and fungal tRNA methyltransferases.

All organisms modify their tRNAs by use of evolutionarily conserved enzymes. Members of the Archaea contain an extensive set of modified nucleotides that were early evidence of the fundamental evolutionary divergence of the Archaea from Bacteria and Eucarya. However, the enzymes responsible for these posttranscriptional modifications were largely unknown before the advent of genome sequencing.

Yeast mitochondrial initiator tRNA is methylated at guanosine 37 by the Trm5-encoded tRNA (guanine-N1-)-methyltransferase.

The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m(1)G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m(1)G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons.

Characterization of the C2 subdomain of yeast mitochondrial initiation factor 2.

The COOH-terminal part of the yeast mitochondrial initiation factor 2 (ymIF2), containing the C2 subdomain, was expressed and purified as a histidine-tagged polypeptide of 137 amino acids. Like the recombinant full-length protein, the C2 subdomain binds both formyl-Met-tRNA(f)(Met) and unformylated Met-tRNA(f)(Met) with only a small preference for the former species. Formation of a binary complex between the C2 subdomain or the full-length ymIF2 and initiator tRNA was also assessed by fluorescence measurements.

Mammalian mitochondrial initiation factor 2 supports yeast mitochondrial translation without formylated initiator tRNA.

Initiation of protein synthesis in mitochondria and chloroplasts is widely believed to require a formylated initiator methionyl-tRNA (fMet-tRNAfMet) in a process involving initiation factor 2 (IF2). However, yeast strains disrupted at the FMT1 locus, encoding mitochondrial methionyl-tRNA formyltransferase, lack detectable fMet-tRNAfMet but exhibit normal mitochondrial function as evidenced by normal growth on non-fermentable carbon sources. Here we show that mitochondrial translation products in Saccharomyces cerevisiae were synthesized in the absence of formylated initiator tRNA.

Purification and characterization of yeast mitochondrial initiation factor 2.

Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E.

The molecular chaperone DnaK is not recruited to translating ribosomes that lack trigger factor.

The molecular chaperone DnaK and trigger factor (TF), a ribosome-associated protein with folding activity, have been implicated in assisting nascent polypeptides to acquire a three-dimensional structure on Escherichia coli ribosomes. We asked whether ribosomes that lack trigger factor would recruit DnaK for synthesis and folding of nascent peptides. For these analyses, translating ribosomes with a homogeneous population of nascent peptides were isolated.

Single synonymous codon substitution eliminates pausing during chloramphenicol acetyl transferase synthesis on Escherichia coli ribosomes in vitro.

The coding sequence for chloramphenicol acetyl transferase (CAT) contains several rare codons; three of them are ATA encoding isoleucine in positions 13, 84 and 119 of the amino acid sequence. Expression of CAT on Escherichia coli ribosomes in vitro results in mostly full-length product but also distinct smaller polypeptides from less than 3 kDa to over 20 kDa. As reported earlier, the smaller polypeptides are the predominant products, if translation is initiated with fluorophore-Met-tRNA(f).