Mechanisms of translational initiation and elongation in mammalian mitochondria.

摘要

Initiation of protein synthesis in mammalian mitochondria involves two protein factors: initiation factor 2 (IF2mt) and initiation factor 3 (IF3mt). Mutations were designed in various domains of IF3 mt to identify the functionally important regions. Residues 170-171, and 175 in the C-terminal domain nearly abolished activity in initiation complex formation and in 55S ribosomal dissociation. However, these mutated proteins bound to the 28S ribosomal subunit with Kd values similar to the wild-type factor. The results suggest that IF3mt plays an active role in initiation of translation.;Mammalian mitochondrial mRNAs have few or no nucleotides prior to the 5' start codon. Both the mammalian mitochondrial 55S ribosome and 28S subunit preferentially formed initiation complexes at a 5' terminal AUG codon over an internal AUG. The selection of the 5' AUG depended on the presence of fMet-tRNA and was enhanced by the presence of IF2mt. Addition of even a few nucleotides 5' to the AUG codon significantly reduced the efficiency of translation. In addition, very few initiation complexes could form on a hybrid mRNA construct consisting of the tRNAMet attached at the 5' end to a mitochondrial protein coding sequence. This observation demonstrates that post-transcriptional processing must occur prior to translation in mammalian mitochondria.;Mutations in the nuclear genes for mammalian mitochondrial translational elongation factors are generally lethal shortly after birth. A mutation of EF-Tsmt (R325W) resulted in a significant reduction in the ability of EF-Tsmt to bind EF-Tumt. A mutation of EF-Tu mt (R336Q) causes infantile encephalopathy arising from defects in mitochondrial translation. The R336Q mutation caused a two-fold decrease ternary complex formation with E. coli aminoacyl-tRNA but completely inactivated EF-Tumt for binding to mitochondrial aminoacyl-tRNA.;Polyamines are important in both prokaryotic and eukaryotic translational systems. Spermine stimulated fMet-tRNA binding to mammalian mitochondrial 55S ribosomes in a manner independent of the identity of the mRNA and to the same degree at all concentrations of IF2mt and IF3mt. The major effect of spermine in promoting initiation complex formation appears to be on the interaction of fMet-tRNA with the ribosome.