Identification of the cap binding domain of human recombinant eukaryotic protein synthesis initiation factor 4E using a photoaffinity analogue
Open Access
- 1 January 1997
- journal article
- research article
- Published by Wiley in Protein Science
- Vol. 6 (1) , 125-131
- https://doi.org/10.1002/pro.5560060114
Abstract
Binding of eIF-4E to the 5′ m7G cap structure of eukaryotic mRNA signals the initiation of protein synthesis. In order to investigate the moiecular basis for this recognition, photoaffinity labeling with [γ-32P]8-N3GTP was used in binding site studies of human recombinant cap binding protein reIF-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 [γ-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 reIF-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 [γ-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.Keywords
Funding Information
- National Science Foundation ((GER-9023681 and MCB 9303661))
- American Heart Association
- National Institutes of Health ((NIH-CA63640))
- National Science Foundation pre-doctoral fellowship (DGE-9553549)
- Glaxo-Wellcome Co.
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