The interaction of mitochondrial translational initiation factor 2 with the small ribosomal subunit

Bovine mitochondrial translational initiation factor 2 (IF-2_mt) 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-2_mt were prepared and tested for their abilities to bind the small (28S) subunit of the mitochondrial ribosome. The binding of wild-type IF-2_mt was strong (K_d∼10-20 nM) and was not affected by fMet-tRNA. Deletion of the N-terminal domain substantially reduced the binding of IF-2_mt 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-2_mt can stabilize the binding of this factor to 28S subunits. No binding was observed for IF-2_mt variants lacking the G-domain which probably plays a critical role in organizing the structure of IF-2_mt. IF-2_mt 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.