Cooperative modulation by eIF4G of eIF4E-binding to the mRNA 5' cap in yeast involves a site partially shared by p20.
Interaction between the mRNA 5'-cap-binding protein eIF4E and the multiadaptor protein eIF4G has been demonstrated in all eukaryotic translation assemblies examined so far. This study uses immunological, genetic 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 mutations in these surface regions disrupts eIF4E-eIF4G association, and thereby polysome formation and growth. The residues at these positions in wild-type eIF4E mediate positive cooperativity between the binding of eIF4G to eIF4E and the latter's cap-affinity. Moreover, two of the mutations confer temperature sensitivity in eIF4G binding to eIF4E which correlates 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 times lower binding affinity than eIF4G for eIF4E. Investigation of a second class of eIF4E mutations reveals that p20 shares only part of eIF4G's binding site on the cap-binding protein. The results presented provide a basis for understanding how cycling of eIF4E and eIF4G occurs in yeast translation and explains how p20 can act as a fine, but not as a coarse, regulator of protein synthesis.
Posttranscriptional Control Group, Department of Biomolecular Sciences, UMIST, P.O. Box 88, Manchester M60 1QD, UK.
M
M
Ptushkina
T
T
von der Haar
S
S
Vasilescu
R
R
Frank
R
R
Birkenhäger
J E
JE
McCarthy
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D008958:Models, Molecular; D008969:Molecular Sequence Data; D039502:Nuclear Cap-Binding Protein Complex; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012315:RNA Caps; D012331:RNA, Fungal / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D000911:Antibodies, Monoclonal; C100383:CBC2 protein, S cerevisiae; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D039502:Nuclear Cap-Binding Protein Complex; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D012315:RNA Caps; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins