The 5-cap and the poly(A) tail act synergistically to increase the translational efficiency of eukaryotic mRNAs, which suggests that these two mRNA elements communicate during translation. We report here that the cap-associated eukaryotic initiation factors (eIFs), i.e. the two isoforms of the cap-binding complex (eIF-4F and eIF-iso4F) and eIF-4B, bind to the poly(A)-binding protein (PABP) both in the presence and absence of poly(A) RNA. The interactions between PABP and eIF-4F, eIF-iso4F, and eIF-4B were measured in the absence of poly(A) RNA using far Western analysis and confirmed by direct fluorescence titration studies. The functional consequence of the interaction between these initiation factors and PABP was examined using RNA binding assays and RNA mobility shift analysis. eIF-4F, eIF-iso4F, and eIF-4B promoted PABP activity through a shift in its equilibrium affinity for poly(A). eIF-iso4G, the large subunit of eIF-iso4F, was the subunit responsible for the interaction between eIF-iso4F and PABP and was the subunit that promoted PABP RNA binding activity. Truncation analysis of eIF-iso4G indicated that a domain close to its N-terminal end appeared to be involved in binding PABP. These results suggest that the interaction between PABP and eIF-4B and eIF-iso4G may be involved in mediating the functional co-dependence observed between the cap and the poly(A) tail during translation.Most eukaryotic mRNAs contain a cap (m 7

Eukaryotic translation initiation factor eIF-4E plays a central role in the recognition of the 7-methylguanosine-containing cap structure of mRNA and the formation of initiation complexes during protein synthesis. eIF-4E exists in both phosphorylated and nonphosphorylated forms, and the primary site of phosphorylation has been identified. Previous studies have suggested that eIF-4E phosphorylation facilitates its participation in protein synthesis. However, the biochemical basis for the functional difference between the two forms of eIF-4E is unknown. To address this directly, we have developed a method for the separation of phosphorylated and nonphosphorylated eIF-4E from rabbit reticulocytes by chromatography on rRNA-Sepharose. Using the resultant purified forms, we have studied the protein's interaction with the cap analogs m7GTP and m7GpppG and with the cap of globin mRNA by fluorescence quenching of tryptophan residues. It was found that phosphorylated eIF-4E had 3-to 4-fold greater affinity for cap analogs and mRNA than nonphosphorylated eIF-4E. The equilibrium binding constants (x 105, expressed as M-1) for the interaction of phosphorylated eIF-4E with m7GTP, m7GpppG, and globin mRNA were 20.0 ± 0.1, 16.4 ± 0.1, and 31.0 ± 0.1, respectively, whereas those for the nonphosphorylated form were 5.5 ± 0.4, 4.3 ± 0.4, and 10.0 ± 0.1, respectively. Treatment with potato acid phosphatase converted the phosphorylated form to the nonphosphorylated form and decreased the binding constant for m7GTP by a factor of3. The increased affinity for mRNA caps may account for the in vivo and in vitro correlations between eIF-4E phosphorylation and accelerated protein synthesis and cell growth.Except in unusual circumstances such as virus infection, cellular stress, or deprivation of nutrients, the overall rate of protein synthesis in eukaryotic cells is thought to be determined by the rate of formation of 48S initiation complexes. This is based on the observations that 48S complexes are present at very low steady-state levels compared with other initiation complexes (1), usually being detected only in the presence of inhibitors (2), and that the ATP-dependent step ofinitiation is rate-limiting (3). Formation ofthe 48S initiation complex involves the binding of mRNA to the 43S initiation complex and is catalyzed by group 4 initiation factors (reviewed in refs. 4-6). The four polypeptides of the eIF-4 group-eIF-4A, eIF-4B, eIF-4E, and eIF-41-collectively bind to the m7GTP-containing cap, unwind mRNA secondary structure at the expense of ATP, and facilitate the movement of the 40S ribosomal subunit along the mRNA until the initiation codon is reached. Various complexes of the eIF-4 polypeptides have been isolated, the best studied of which is termed eIF-4F. eIF-4F isolated from mammalian cells was originally shown to contain the polypeptides eIF-4A, eIF-4E, and eIF-4y (7, 8), but other purifications yield a twocomponent factor consisting of eIF-4E and eIF-4y (9-11). Similarly, eIF-4F and eIF(iso)-4F from plants (12) and ...

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