COOPERATIVE MODULATION BY EIF4G OF EIF4E-BINDING TO THE MESSENGER-RNA5'-CAP IN YEAST INVOLVES A SITE PARTIALLY SHARED BY P20

Interaction between the mRNA 5'-cap-binding protein eIF4E and the mult iadaptor protein eIF4G has been demonstrated in all eukaryotic transla tion assemblies examined so far, This study uses immunological, geneti c and biochemical methods to map the surface amino acids on eIF4E that contribute to eIF4G binding. Cap-analogue chromatography and surface plasmon resonance (SPR) analyses demonstrate that one class of mutatio ns in these surface regions disrupts eIF4E eIF4G association, and ther eby polysome formation and growth. The residues at these positions in wildtype eIF4E mediate positive cooperativity between the binding of e IF4G to eIF4E and the latter's cap-affinity. Moreover, two of the muta tions confer temperature sensitivity in eIF4G binding to eIF4E which c orrelates with the formation of large numbers of inactive ribosome 80S couples in vivo and the loss of cellular protein synthesis activity. The yeast 4E-binding protein p20 is estimated by SPR to have a ten tim es lower binding affinity than eIF4G for eIF4E, Investigation of a sec ond class of eIF4E mutations reveals that p20 shares only part of eIF4 G's binding site on the cap-binding protein. The results presented pro vide a basis for understanding how cycling of eIF4E and eIF4G occurs i n yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.