Photoaffinity labeling of the cap-binding protein complex with ATP/dATP. Differential labeling of free eukaryotic initiation factor 4A and the eukaryotic initiation factor 4A component of the cap-binding protein complex with [alpha-32P]ATP/dATP.
It has been suggested that the cap-binding protein complex is involved in ATP-mediated melting of 5'-mRNA secondary structure to facilitate ribosome binding during initiation of translation in eukaryotic cells (Edery, I., Lee, K. A. W., and Sonenberg, N. (1984) Biochemistry 23, 2456-2462). Consequently, we have studied the interaction of dATP/ATP with the eukaryotic cap-binding protein complex by UV photoaffinity labeling. UV irradiation of the cap-binding protein complex in the presence of [alpha-32P]dATP/ATP resulted in the cross-linking of this compound to the 50-kDa polypeptide of the complex. This polypeptide is almost identical to the previously characterized eukaryotic initiation factor (eIF) 4A. We examined the ability of dATP/ATP to cross-link to eIF-4A and found that it cross-links less efficiently (approximately 60-fold on a molar basis) compared to the cross-linking obtained for the eIF-4A component of the cap-binding protein complex. Irradiation of purified eIF-4A together with the cap-binding protein complex in the presence of [alpha-32P]dATP resulted in greater than additive labeling of the eIF-4A component of the cap-binding protein complex and purified eIF-4A, suggesting a synergistic interaction between purified eIF-4A, the cap-binding protein complex, and dATP/ATP. We also report that photoaffinity labeling of eIF-4A and the eIF-4A component in the cap-binding protein complex is stimulated by eIF-4B, but not by other initiation factors or mRNA.
D000255:Adenosine Triphosphate; D000345:Affinity Labels; D000818:Animals; D002352:Carrier Proteins; D003838:Deoxyadenine Nucleotides; D039601:Eukaryotic Initiation Factor-4A; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D010448:Peptide Initiation Factors; D010761:Phosphorus Radioisotopes; D010777:Photochemistry; D039381:RNA Cap-Binding Proteins; D011817:Rabbits
D000345:Affinity Labels; D002352:Carrier Proteins; D003838:Deoxyadenine Nucleotides; D010448:Peptide Initiation Factors; D010761:Phosphorus Radioisotopes; D039381:RNA Cap-Binding Proteins; D000255:Adenosine Triphosphate; D039601:Eukaryotic Initiation Factor-4A; D008274:Magnesium; C026600:2'-deoxyadenosine triphosphate