Roles of the N- and C-Terminal Domains of Mammalian Mitochondrial Initiation Factor 3 in Protein Biosynthesis

• Published in: Journal of molecular biology 2008-12, Vol.384 (4), p.929-940
• Main Authors: Haque, Md. Emdadul, Spremulli, Linda L.
• Format: Article
• Language: English
• Subjects: Animals; Escherichia coli; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Eukaryotic Initiation Factors - genetics; Eukaryotic Initiation Factors - metabolism; initiation complex; initiation factor 3; Kinetics; mitochondria; Prokaryotic Initiation Factor-3 - genetics; Prokaryotic Initiation Factor-3 - metabolism; Protein Binding; Protein Biosynthesis; Protein Structure, Tertiary; protein synthesis; ribosome; Ribosome Subunits, Small - metabolism
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2008.09.077

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.