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. Based on 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 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 247–248 in the C-terminal extension abolished the ability of IF3mt to reduce the binding of fMet-tRNA to the ribosome in the absence of mRNA. The results from this paper suggest that IF3mt plays an active role in initiation of translation.
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