Mitochondrial translational initiation factor 3 (IF3mt) is a 29 kDa protein that has N- and C-terminal domains, homologous to prokaryotic IF3, connected by a linker region. The homology domains are preceded and followed by short extensions. No information is currently available on the specific residues in IF3mt important for its activity. On the basis of homology models of IF3mt, mutations were designed in the N-terminal, C-terminal, and linker domains to identify the functionally important regions. Mutation of residues 170−171, and 175 in the C-terminal domain to alanine resulted in a nearly complete loss of activity in initiation complex formation and in the dissociation of mitochondrial 55S ribosomes. However, these mutated proteins bind to the small (28S) subunit of the mammalian mitochondrial ribosome with Kd values similar to that of the wild-type factor. These mutations appear to lead to a factor defective in the ability to displace the large (39S) subunit of the ribosome from the 55S monosomes in an active process. Other mutations in the N-terminal domain, the linker region, and the C-terminal domain had little or no effect on the ability of IF3mt to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 247 and 248 in the C-terminal extension abolished the ability of IF3mt to reduce the level of binding of fMet-tRNA to the ribosome in the absence of mRNA. Our results suggest that IF3mt plays an active role in initiation of translation.

Bovine mitochondrial translational initiation factor 2 (IF-2mt) is organized into four domains, an N-terminal domain, a central G-domain and two C-terminal domains. These domains correspond to domains III–VI in the six-domain model of Escherichia coli IF-2. Variants in IF-2mt were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2mt was strong (Kd∼10–20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2mt to 28S subunits. However, the addition of fMet-tRNA stimulated the binding of this variant at least 2-fold demonstrating that contacts between fMet-tRNA and IF-2mt can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2mt variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2mt. IF-2mt contains a 37-amino acid insertion region between domains V and VI that is not found in the prokaryotic factors. Mutations in this region caused a significant reduction in the ability of the factor to promote initiation complex formation and to bind 28S subunits.

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.Highlights► Mitochondrial protein synthesis is a critical component of the oxidative phosphorylation system. ► A biochemical perspective on mitochondrial protein synthesis is provided. ► Mutations in this translational systems lead to human disease.

Mitochondria possess two elongation factor Gs: one with translocation activity (EF-G1mt) and the other with no confirmed activity (EF-G2mt). Tsuboi et al. (2009) now show that the function of EF-G2mt is not in elongation but, rather, in ribosome recycling.

Bacterial initiation factor 3 (IF3) is organized into N- and C-domains separated by a linker. Mitochondrial IF3 (IF3mt) has a similar domain organization, although both domains have extensions not found in the bacterial factors. Constructs of the N- and C-domains of IF3mt with and without the connecting linker were prepared. The Kd values for the binding of full-length IF3mt and its C-domain with and without the linker to mitochondrial 28S subunits are 30, 60, and 95 nM, respectively, indicating that much of the ribosome binding interactions are mediated by the C-domain. However, the N-domain binds to 28S subunits with only a 10-fold lower affinity than full-length IF3mt. This observation indicates that the N-domain of IF3mt has significant contacts with the protein-rich small subunit of mammalian mitochondrial ribosomes. The linker also plays a role in modulating the interactions between the 28S subunit and the factor; it is not just a physical connector between the two domains. The presence of the two domains and the linker may optimize the overall affinity of IF3mt for the ribosome. These results are in sharp contrast to observations with Escherichia coli IF3. Removal of the N-domain drastically reduces the activity of IF3mt in the dissociation of mitochondrial 55S ribosomes, although the C-domain itself retains some activity. This residual activity depends significantly on the linker region. The N-domain alone has no effect on the dissociation of ribosomes. Full-length IF3mt reduces the binding of fMet–tRNA to the 28S subunit in the absence of mRNA. Both the C-terminal extension and the linker are required for this effect. IF3mt promotes the formation of a binary complex between IF2mt and fMet–tRNA that may play an important role in mitochondrial protein synthesis. Both domains play a role promoting the formation of this complex.