Exploration of the roles of IF3 mt in mammalian mitochondrial translation

The initiation of mammalian mitochondrial protein synthesis is poorly understood. Two initiation factors with homology to bacterial IF2 and IF3 have been identified. IF3 mt is a 29 kDa protein with an internal region having homology to prokaryotic IF3. This homology region consists of an N‐terminal domain, a linker and a C‐terminal domain. The homology domains are preceded and followed by short extensions. No information is currently available on the regions of IF3 mt important for its activity. Based on homology models of IF3 mt , mutations were designed in the N‐terminal, C‐terminal, and linker domains to identify the functions of these regions. Mutation of residues 66–70 and 121–124 in the N‐terminal domain and residues 194–197 in the C‐terminal domain had no effect on the ability of IF3 to promote initiation complex formation on mitochondrial 55S ribosomes. Mutation of residues 143–147 in the linker region and residues 184–186 in the C‐terminal domain caused a significant reduction in activity indicating an important role for this region for initiation complex formation. Mutation of histidine 170 and aspartic acid 171 to alanine caused a nearly complete loss of activity. However, sucrose density centrifugation indicates that this variant can bind to 28S subunits. Mutation of the nearby region (residues 175–177) also resulted in a significant loss of activity. The causes for these defects are under investigation.