Assays for eukaryotic translation factors that bind mRNA.

After a brief introduction to the function of the mRNA-specific translation factors eIF4A, eIF4B, and EIF4F, this article presents appropriate methodology for the study of the translation factors associated with the activation of mRNA for translation in eukaryotic systems. The purification of eIF4A, eIF4B, and eIF4F from rabbit reticulocyte lysates is given along with a procedure for the purification of hemoglobin mRNA. These purifications provide reagents for the model assays of RNA binding (as retention on nitrocellulose filters) and RNA-dependent ATP hydrolysis. With additional reagents available as RNA transcripts using either T7 or SP6 polymerase, two additional assays are possible, crosslinking to the oxidized cap of the mRNA or the ATP-dependent reaction of RNA unwinding (helicase assay). Finally, there is a description of the most biological assay for the utilization of natural mRNAs, the synthesis of the authentic polypeptide chain, in this instance hemoglobin. Throughout the portion of this article that deals with the biological assays, helpful hints are provided to ensure that the assay works, and suggestions are provided for control experiments to ensure that an artifact is not being studied. It is hoped that this information will facilitate the study of either the regulation of factor activity (for eIF4A, eIF4B, or eIF4F) or the translation efficiency (sometimes regulated) of various mRNAs.