Mapping Functional Regions of Mitochondrial Initiation Factor 3

The initiation of mammalian mitochondrial protein synthesis is poorly understood. Two initiation factors with homology to bacterial IF2 and IF3 have been identified. IF3mt is a 29 kDa protein with an internal region of 183 amino acids with homology to prokaryotic IF3. This homology region consists of an N‐terminal domain, a linker and a C‐terminal domain. The N‐terminal homology domain is preceded by an extension of 31 amino acids, and the C‐terminal domain is followed by an extension of 33 amino acids. In prokaryotes, the C‐terminal domain is thought to be responsible for most of the activities of IF3. No information is currently available about the regions of IF3mt important for its activity. Based on homology models of IF3mt, mutations were designed in the N‐terminal, C‐terminal, and linker domains to help identify the functions of these regions. In general, charged residues in surface regions predicted to be in contact with ribosomes were changed to alanine residues. When tested on E. coli ribosomes, mutations of residues 121–124 in the N‐terminal domain had no effect on activity. Mutation of residues 143–147 in the linker region caused a 35% reduction in activity, and two mutations in the C‐terminal domain caused 30% (residues 184–186) and nearly complete (residues 170–171) reductions in activity. These mutated proteins will be tested for activity on mitochondrial 55S ribosomes. In addition, their ability to bind to mitochondrial 28S subunits will be determined. Research supported by NIH grant #GM032734