Evidence for an Active Role of IF3mt in the Initiation of Translation in Mammalian Mitochondria

摘要

ABSTRACT: Mitochondrial translational initiation factor 3 (IF3mt) is a 29 kDa protein that has N- andnC-terminal domains, homologous to prokaryotic IF3, connected by a linker region. The homology domainsnare preceded and followed by short extensions. No information is currently available on the specific residuesnin IF3mt important for its activity. On the basis of homology models of IF3mt, mutations were designed innthe N-terminal, C-terminal, and linker domains to identify the functionally important regions. Mutationnof residues 170-171, and 175 in the C-terminal domain to alanine resulted in a nearly complete loss ofnactivity in initiation complex formation and in the dissociation of mitochondrial 55S ribosomes. However,nthese mutated proteins bind to the small (28S) subunit of the mammalian mitochondrial ribosome with Kdnvalues similar to that of the wild-type factor. These mutations appear to lead to a factor defective in thenability to displace the large (39S) subunit of the ribosome from the 55S monosomes in an active process.nOther mutations in the N-terminal domain, the linker region, and the C-terminal domain had little or noneffect on the ability of IF3mt to promote initiation complex formation on mitochondrial 55S ribosomes.nMutation of residues 247 and 248 in the C-terminal extension abolished the ability of IF3mt to reduce thenlevel of binding of fMet-tRNA to the ribosome in the absence of mRNA. Our results suggest that IF3mtnplays an active role in initiation of translation.