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 m(7)GpppN 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 eIF4E were fused to the RNA-binding domain of the lambda phage antiterminator protein N to generate the chimeric proteins lambda 4A, lambda 4E-(102) (abolished cap binding), and lambda 4E-(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 lambda -N binding site (boxB) in the intercistronic space. We show that lambda 4E-(102), but neither the double mutant lambda 4E-(73-102) nor lambda 4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all lambda -fusion proteins are capable of interacting with the appropriate endogenous eIF4F 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, lambda 4A 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.