Expression, purification, and mechanistic studies of bovine mitochondrial translational initiation factor 2.
A complete cDNA clone encoding bovine mitochondrial translational initiation factor 2 (IF-2mt) has been obtained. The regions of the cDNA corresponding to mature IF-2mt and several of its functional domains have been expressed in Escherichia coli as histidine-tagged proteins. The precursor (approximately 90 kDa) and mature (approximately 85 kDa) forms of IF-2mt are toxic to E. coli and can only be expressed at low levels. Shorter forms of this factor (approximately 80 and approximately 72 kDa) are also found during the expression of mature IF-2mt. The various forms of IF-2mt can be separated by high performance liquid chromatography. 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-2mt can bind to mitochondrial ribosomes in the absence of GTP, initiator tRNA, or messenger RNA. The presence of GTP stimulates IF-2mt binding to ribosomes about 3-fold. IF-2mt interacts only weakly with GTP or with the initiator tRNA in the absence of ribosomes. Molecular dissection of IF-2mt shows that a long deletion (approximately 150 amino acid residues) from the NH2-terminal region does not affect its activity in vitro. The COOH domain of IF-2mt (amino acid residues 332-727) can bind to ribosomes even though it does not promote initiator-tRNA binding.
Ma
J
J
Department of Chemistry and Lineberger Comprehensive Cancer Research Center, University of North Carolina, Chapel Hill, 27599-3290, USA.
Spremulli
L L
LL
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010448:Peptide Initiation Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D016324:Sequence Tagged Sites
D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D010448:Peptide Initiation Factors; D039881:Prokaryotic Initiation Factor-2; D012358:RNA, Transfer, Met; D011994:Recombinant Proteins; C024988:tRNA, formylmethionine-