Dominant negative mutants of mammalian translation initiation factor eIF‐4A define a critical role for eIF‐4F in cap‐dependent and cap‐independent initiation of translation.

Eukaryotic translation initiation factor‐4A (eIF‐4A) plays a critical role in binding of eukaryotic mRNAs to ribosomes. It has been biochemically characterized as an RNA‐dependent ATPase and RNA helicase and is a prototype for a growing family of putative RNA helicases termed the DEAD box family. It is required for mRNA‐ribosome binding both in its free form and as a subunit of the cap binding protein complex, eIF‐4F. To gain further understanding into the mechanism of action of eIF‐4A in mRNA‐ribosome binding, defective eIF‐4A mutants were tested for their abilities to function in a dominant negative manner in a rabbit reticulocyte translation system. Several mutants were demonstrated to be potent inhibitors of translation. Addition of mutant eIF‐4A to a rabbit reticulocyte translation system strongly inhibited translation of all mRNAs studied including those translated by a cap‐independent internal initiation mechanism. Addition of eIF‐4A or eIF‐4F relieved inhibition of translation, but eIF‐4F was six times more effective than eIF‐4A, whereas eIF‐4B or other translation factors failed to relieve the inhibition. Kinetic experiments demonstrated that mutant eIF‐4A is defective in recycling through eIF‐4F, thus explaining the dramatic inhibition of translation. Mutant eIF‐4A proteins also inhibited eIF‐4F‐dependent, but not eIF‐4A‐dependent RNA helicase activity. Taken together these results suggest that eIF‐4A functions primarily as a subunit of eIF‐4F, and that singular eIF‐4A is required to recycle through the complex during translation. Surprisingly, eIF‐4F, which binds to the cap structure, appears to be also required for the translation of naturally uncapped mRNAs.