Eukaryotic Translation Initiation Factor 4E (eIF4E) Binding Site and the Middle One-Third of eIF4GI Constitute the Core Domain for Cap-Dependent Translation, and the C-Terminal One-Third Functions as a Modulatory Region

Abstract

The mammalian eukaryotic initiation factor 4GI (eIF4GI) may be divided into three roughly equal regions; an amino-terminal one-third (amino acids [aa] 1 to 634), which contains the poly(A) binding protein (PABP) and eIF4E binding sites; a middle third (aa 635 to 1039), which binds eIF4A and eIF3; and a carboxy-terminal third (aa 1040 to 1560), which harbors a second eIF4A binding site and a docking sequence for the Ser/Thr kinase Mnk1. Previous reports demonstrated that the middle one-third of eIF4GI is sufficient for cap-independent translation. To delineate the eIF4GI core sequence required for cap-dependent translation, various truncated versions of eIF4GI were examined in an in vitro ribosome binding assay with β-globin mRNA. A sequence of 540 aa encompassing aa 550 to 1090, which contains the eIF4E binding site and the middle region of eIF4GI, is the minimal sequence required for cap-dependent translation. In agreement with this, a point mutation in eIF4GI which abolished eIF4A binding in the middle region completely inhibited ribosomal binding. However, the eIF4GI C-terminal third region, which does not have a counterpart in yeast, modulates the activity of the core sequence. When the eIF4A binding site in the C-terminal region of eIF4GI was mutated, ribosome binding was decreased three- to fourfold. These data indicate that the interaction of eIF4A with the middle region of eIF4GI is necessary for translation, whereas the interaction of eIF4A with the C-terminal region plays a modulatory role.

ACKNOWLEDGMENTS

We thank W. C. Merrick for eIF2, eIF3, and eIF4F proteins used for a preliminary experiment, T. Skern for rhinovirus 2A^pro, A. Gradi for the anti-eIF4GI antibody, and R. Fukunaga for the Mnk1 plasmid. We are indebted to C. Lister for excellent technical assistance. We thank B. Raught and A.-C. Gingras for sharing unpublished data and critically reading the manuscript.

S.M. was supported by research fellowships of the Japan Society for the Promotion of Science for Young Scientists. This work was supported by a grant from the Medical Research Council of Canada to N.S. N.S. is a Distinguished Scientist of the Medical Research Council of Canada and a Howard Hughes Medical Institute International Scholar.

ADDENDUM IN PROOF

Since the submission of this paper, a report by De Gregorio et al. (EMBO J. 18:4865–4874, 1999) also defined a conserved central domain (aa 642 to 1091) of eIF4G as an autonomous ribosome recruitment core in vivo.