Tethered-function analysis reveals that elF4E can recruit ribosomes independent of its binding to the cap structure

The cap-binding complex elF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: elF4E, -4G, and -4A. The m⁷GpppN cap-binding subunit elF4E binds the N-terminal region of elF4G, which in turn contacts elF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of elF4G to an mRNA is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865-4874). Here we exploit this approach to assess which of the other subunits of elF4F can exert this function, elF4AI or mutant forms of elF4E were fused to the RNA-binding domain of the λ phage antiterminator protein N to generate the chimeric proteins λ4A, λ4E_-102 (abolished cap binding), and λ4E-_73-102 (impaired binding to both, the cap and elF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific λ-N binding site (boxB) in the intercistronic space. We show that λ4E-₁₀₂, but neither the double mutant λ4E-_73-102 nor λ4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all λ-fusion proteins are capable of interacting with the appropriate endogenous elF4F subunits. These results reveal that elF4E, as well as elF4G, can drive ribosome recruitment independent of a physical link to the cap structure. In spite of its interaction with endogenous elF4G, λ4A does not display this property, elF4A thus appears to supply an essential auxiliary function to elF4F that may require its ability to cycle into and out of this complex.