Purification and characterization of bacterially expressed mammalian translation initiation factor 5 (eIF-5): demonstration that eIF-5 forms a specific complex with eIF-2.
Eukaryotic translation initiation factor 5, eIF-5, has been purified to apparent electrophoretic homogeneity from overproducing Escherichia coli cells expressing the cDNA of the initiation factor under the control of the T7 promoter-T7 RNA polymerase system. Purified recombinant eIF-5 mimics natural eIF-5 isolated from mammalian cells in size, in specific activity, in its ability to catalyze the hydrolysis of GTP bound to the 40S initiation complex, and in the subsequent joining with 60S ribosomal subunits to form the 80S initiation complex. Further characterization of eIF-5 demonstrates that eIF-5 specifically associates with eIF-2, forming an eIF-2.eIF-5 complex. The protein complex sediments in glycerol gradients with an apparent M(r) of 160,000, suggesting that the two proteins associate in a 1:1 stoichiometry. The association between the two initiation factors is highly specific. Addition of 32P-labeled eIF-5 to a partially purified rabbit reticulocyte initiation factor preparation that contained, in addition to eIF-2 and eIF-5, other initiation factors and many other proteins resulted in the specific binding of labeled eIF-5 only to eIF-2, forming a 160-kDa protein complex. In agreement with these observations, we found that in crude initiation factor preparations derived from rabbit reticulocyte lysates, eIF-5 was present as an eIF-2.eIF-5 complex. The significance of eIF-2.eIF-5 complex formation in the overall mechanism of GTP hydrolysis in protein synthesis initiation is discussed.
Chaudhuri
J
J
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461.
Das
K
K
Maitra
U
U
D000818:Animals; D004926:Escherichia coli / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry*; D040061:Eukaryotic Initiation Factor-5; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012156:Reticulocytes / Q000737:chemistry
D015852:Eukaryotic Initiation Factor-2; D040061:Eukaryotic Initiation Factor-5; D010448:Peptide Initiation Factors; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate