Mutations in the S4–H2 loop of eIF4E which increase the affinity for m 7GTP

Abstract

Eukaryotic initiation factor 4E (eIF4E) binds the 5′-cap of eukaryotic mRNAs and overexpression of eIF4E in epithelial cell cancers correlates with the metastases/tissue invasion phenotype. Photolabeling of eIF4E with [γ- 32P]8-azidoguanosine 5′-triphosphate (8-N 3GTP) demonstrated cross-linking at Lys-119 in the S4–H2 loop which is distant from the m 7GTP binding site [Marcotrigiano et al. (1997) Cell 89, 951–961; Friedland et al. (1997) Protein Sci. 6, 125–131]. Modeling studies indicate that 8-N 3GTP cross-linked with Lys-119 because it binds a site that is occupied by the second nucleotide of a bound mRNA. Mutagenesis of the S4–H2 loop produced proteins with a 5–10-fold higher affinity for m 7GTP than wild-type eIF4E. These mutants of eIF4E may have uses in selectively purifying mRNAs with intact 5′-ends or in determining how the promyelocytic leukemia protein decreases the affinity of eIF4E for mRNA caps.