Interaction of protein synthesis initiation factors with the mRNA cap structure

Abstract

The mechanism of mRNA recognition by proteins interacting with the mRNA cap structure was investigated by photochemical cross-linking of proteins with 32P-labelled reoviral RNAs. Using ribosomal washes as a source of eukaryotic protein synthesis initiation factors, we identified the well-known cap binding proteins eIF-4B and -4E, but eIF-2 and eIF-3 as well. The interplay of purified eIF-4A, -4B, and -4F was studied in relation to ATP dependence and cap analogue sensitivity of cap binding. Next to their well-known roles in the initiation process, eIF-2 and eIF-3 also cross-linked to the 5′ cap. eIF-2 stimulated eIF-4B and -4E cross-linking, an observation that has been previously described more extensively. The interaction of eIF-2 with the 5′ end of mRNA was extremely sensitive to K +-ions and was resistant to a high concentration of Mg 2+-ions; this influence of mono- and divalent ions was in contrast with the cross-linking of eIF-4B and 4E. Optimal interaction of these factors was obtained at moderate K + concentration and low Mg 2+-ion concentrations. eIF-2 cross-linking was sensitive to high protein to mRNA ratios indicating a weak affinity as compared to eIF-4E and -4B. The interaction of eIF-3 with the cap of mRNA is also weak as it was counteracted by all other cap binding proteins, leading to an inability to detect the cross-linking of this protein in crude eIF preparations. Time kinetics of formation of complexes suggested eIF-2 to be one of the first factors to interact with mRNA. Preformed RNA-protein complexes were dissociated after cap analogue addition, suggesting reversible interactions between RNA and proteins.