EXPRESSION, PURIFICATION, AND MECHANISTIC STUDIES OF BOVINE MITOCHONDRIAL TRANSLATIONAL INITIATION-FACTOR-2

A complete cDNA clone encoding bovine mitochondrial translational init iation factor 2 (IF-2(mt)) has been obtained. The regions of the cDNA corresponding to mature IF-2(mt) and several of its functional domains have been expressed in Escherichia coli as histidine-tagged proteins. The precursor (similar to 90 kDa) and mature (similar to 85 kDa) form s of IF-2(mt) are toxic to E. coli and can only be expressed at low le vels. Shorter forms of this factor (similar to 80 and similar to 72 kD a) are also found during the expression of mature IF-2(mt). The variou s forms of IF-2(mt) can be separated by high performance liquid chroma tography. All of these forms are active in promoting the GTP-dependent binding of formyl-Met-tRNA to the small subunit of either E. coli or bovine mitochondrial ribosomes. IF-2(mt) can bind to mitochondrial rib osomes in the absence of GTP, initiator tRNA, or messenger RNA. The pr esence of GTP stimulates IF-2(mt) binding to ribosomes about 3-fold. I F-2(mt) interacts only weakly with GTP or with the initiator tRNA in t he absence of ribosomes. Molecular dissection of IF-2(mt) shows that a long deletion (similar to 150 amino acid residues) from the NH2-termi nal region does not affect its activity in vitro. The COOH domain of I F-2(mt) (amino acid residues 332-727) can bind to ribosomes even thoug h it does not promote initiator-tRNA binding.