Cap-binding proteins specifically bind to the 7-methyl guanosine (m 7 G) functional group at the 5 end of eukaryotic mRNAs. A novel Arabidopsis thaliana protein has been identified that has sequence similarity to capbinding proteins but is clearly a different form of the protein. The most obvious primary sequence difference is the substitution of two of the eight conserved tryptophan residues with other aromatic amino acids in the novel protein. Analogous forms of this novel protein appear to be present in other higher eukaryotes but not in yeast. Analysis of the native and recombinant forms of the novel protein by retention on m 7 GTP-Sepharose indicate that it is a functional cap-binding protein. Measurements of the dissociation constant for this protein indicate that it binds m 7 GTP 5-20-fold tighter than eukaryotic initiation factor (eIF)(iso)4E. The novel protein also supports the initiation of translation of capped mRNA in vitro. Biochemical analysis and yeast two-hybrid data indicate that it interacts with eIF(iso)4G to form a complex. Based on these observations, this protein appears to be able to function as a cap-binding protein and is given the designation of novel cap-binding protein (nCBP).Cap-binding proteins (eIF4E) 1 specifically recognize and bind the m 7 G functional group found at the 5Ј end of most eukaryotic cellular mRNAs (for recent reviews, see Refs. 1 and 2). The binding of eIF4E is thought to be the first step in the assembly of several initiation factors on the mRNA prior to the binding of the 40 S ribosome (1-3). One of the structural features of all eIF4E proteins is the presence of eight tryptophan residues in the same relative positions. Recently, the cocrystal structures of the mouse eIF4E (4) and yeast eIF4E (5) with m 7 GDP were solved. Tryptophan residues 3, 5, and 8 were found to be involved in binding the m 7 G functional group (4,5). eIF4E is usually associated with a larger subunit (eIF4G) in a complex named eIF4F (1, 6). eIF4A may also be present in the eIF4E⅐eIF4G complex depending upon the method of purification (7,8). Higher plants possess a unique second form of eIF4F designated eIF(iso)4F (9). eIF(iso)4F contains distinct forms of the cap-binding protein (eIF(iso)4E) and the larger subunit (eIF(iso)4G) (10). The two isoenzyme forms of plant eIF4F have the same activities in vitro (9); however, the eIF(iso)4E prefers hypermethylated caps and mRNAs with less secondary structure (11,12). Recently, a second form of mammalian eIF4G was reported (13). However, this form of eIF4G was not reported to have a distinct form of eIF4E associated with it and does not appear to be the functional equivalent of eIF(iso)4G.The mammalian eIF4E is known to be phosphorylated at Ser-209, and the phosphorylation state appears to correlate with activity of the protein (reviewed in Refs. 14 and 15). Certain stimuli, including insulin and several growth factors, induce phosphorylation of eIF4E (14). Overexpression of the mammalian eIF4E results in cell transformation, suggesting that eIF4E le...

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