Specificity and functional role of mammalian mitochondrial translational initiation factor 3 in initiation of protein synthesis. A comprehensive study of C and N domains with and without linker

Mammalian mitochondrial initiation factor 3 (IF3 mt ) has a central region with homology to the bacterial factor. This homology region is preceded by an N‐terminal extension and followed by C‐terminal extension. The role of these extensions on the binding of IF3 mt to mitochondrial 28S subunit has been studied using a microcon centrifugation assay followed by Western bloting. The K D for the binding of the full length IF3 mt to 28S subunit is 30 nM. Removal of the extensions at the N‐ and C‐termini has no effect on this value implying that the major determinants for ribosomal subunit binding by IF3 mt lie within the region homologous to the bacterial factor. No binding is observed to the 39S large subunit or 55S monosomes as determined by sucrose density gradient centrifugation. The region of IF3 mt homologous to E. coli IF3 can be divided into 3 sections, an N‐terminal domain, a flexible linker and a C‐terminal domain. The flexible linker joins to domains. Four truncated derivatives of IF3 mt were prepared covering the N‐terminal extension and N‐terminal homology domain with and without linker, and the C‐terminal homology domain and C‐terminal extension with and without the linker. The C‐terminal region of IF3 mt with the linker and without the linker is 4‐fold and 2‐fold, respectively, more active than full length IF3 mt in promoting initiation complex formation on E. coli ribosomes in the presence of fMet‐tRNA, poly(A,U,G) and IF2 mt . (Supported by NIH Grant GM32734)