Identification of the cap binding domain of human recombinant eukaryotic protein synthesis initiation factor 4E using a photoaffinity analogue.
Binding of eIF-4E to the 5' m7G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In order to investigate the molecular basis for this recognition, photoaffinity labeling with [gamma-32P]8-N3GTP was used in binding site studies of human recombinant cap binding protein eIF-4E. Competitive inhibition of this cap analogue by m7GTP and capped mRNA indicated probe specificity for interaction at the protein binding site. Saturation of the binding site with [gamma-32P]8-N3GTP further demonstrated the selectivity of photoinsertion. Aluminum (III)-chelate chromatography and reverse-phase HPLC were used to isolate the binding site peptide resulting from digestion of photolabeled eIF-4E with modified trypsin. Amino acid sequencing identified the binding domain as the region containing the sequence Trp 113-Arg 122.Lys 119 was not identified in sequencing analysis nor was it cleaved by trypsin. These results indicate that Lys 119 is the residue directly modified by photoinsertion of [gamma-32P]8-N3GTP. A detailed understanding of eIF-4E.m7G mRNA cap interactions may lead the way to regulating this essential protein-RNA interaction for specific mRNA in vivo.
Friedland
D E
DE
Department of Chemistry, Hunter College of the City University of New York, New York 10021, USA.
Shoemaker
M T
MT
Xie
Y
Y
Wang
Y
Y
Hagedorn
C H
CH
Goss
D J
DJ
D000345:Affinity Labels; D000595:Amino Acid Sequence; D039561:Eukaryotic Initiation Factor-4E; D006801:Humans; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D000345:Affinity Labels; D039561:Eukaryotic Initiation Factor-4E; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins