Characterization of eukaryotic initiation factor 4A, a protein involved in ATP-dependent binding of globin mRNA.
Eukaryotic initiation factor 4A (eIF-4A) has been purified (to apparent homogeneity) from rabbit reticulocyte lysate. It is a single polypeptide accounting for at least 90% of the Coomassie blue staining material when subjected to gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight was determined by gel electrophoresis under denaturing conditions at two different bisacrylamide to acrylamide ratios, by gel filtration under native conditions and by sedimentation equilibrium at three different protein concentrations. Additional physical properties of the polypeptide were also determined. In an attempt to characterize the function of eIF-4A, a protein specifically required for mRNA translation, an assay was developed which measures the protein-dependent retention of radiolabeled hemoglobin mRNA on nitrocellulose filters. These studies led to the discovery of an ATP-stimulated binding of mRNA which is dependent on the presence of eIF-4A and eIF-4B that also contains the 24,000-dalton cap binding protein. The reaction apparently requires ATP hydrolysis since a nonhydrolyzable analogue of ATP, adenosine 5'-(beta, gamma-imino)triphosphate, does not stimulate mRNA binding and GTP cannot substitute for ATP. In addition, ATP-stimulated binding of mRNA can be inhibited by an analog of the mRNA 5' terminus, m7GMP, suggesting recognition of the capped 5' end of hemoglobin mRNA. Consistent with this suggestion, ATP also stimulated the covalent cross-linking of eIF-4A and eIF-4B to the cap of oxidized reovirus mRNA, an interaction that was inhibited by m7GDP.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D039601:Eukaryotic Initiation Factor-4A; D039642:Eukaryotic Initiation Factors; D005914:Globins / Q000235:genetics*; D006454:Hemoglobins / Q000235:genetics; D008970:Molecular Weight; D010448:Peptide Initiation Factors / Q000302:isolation & purification / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D000596:Amino Acids; D039642:Eukaryotic Initiation Factors; D006454:Hemoglobins; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; C031799:eIF-4B; D000255:Adenosine Triphosphate; D005914:Globins; D039601:Eukaryotic Initiation Factor-4A