Role of the insertion region in mammalian mitochondrial initiation factor 2 in translational initiation

Prokaryotic protein synthesis requires three essential initiation factors (IFs). IF2 promotes the binding of the initiator tRNA to the small subunit of the ribosome. IF1 prevents incorrect initiation and stimulates the activities of IF2 and IF3. The mammalian mitochondrial translation initiation system is known to have two of the traditional translation initiation factors, IF2mt and IF3mt. No mitochondrial homolog of IF1 has been detected biochemically or by database searching. IF2mt has an insertion of 37 amino acids, when aligned with E. coli IF2, that is modeled to interact with the mitochondrial ribosome in the same region where IF1 is known to associate in the prokaryotic ribosome. This observation lead to the idea that the insertion might play the role of IF1 in the mitochondrial system. To study this possibility the insertion in IF2mt was removed and replaced with the truncated homologous region from E. coli IF2 (IF2mt{\Delta}eco). The IF2mt insertion was also incorporated into E. coli IF2 in the same manner as above (IF2eco{ \Delta }mt). When tested on E. coli ribosomes IF2mt{\Delta}eco was about 20 % as active in promoting $[^{35}S]$fMet-tRNA incorporation as IF2mt when tested in the presence of IF1. IF2mt{\Delta}eco was about 30 % as active as IF2mt when assayed without IF1. As predicted, IF1 stimulated initiation complex formation on prokaryotic ribosomes with E. coli IF2 and IF2mt{\Delta}eco, 5 and 4 fold, respectively, but did not stimulate IF2mt whose insertion plays the role of IF1