Eukaryotic initiation factor 4A is the component that interacts with ATP in protein chain initiation.

Abstract

Protein synthesis in a resolved homogenate of wheat germ requires ATP and 8 factors functioning at the level of protein chain initiation. To identify the component(s) interacting with ATP, the different factors were treated with the ATP affinity analog 5''-p-fluorosulfonylbenzoyladenosine (FSBA) and tested for their function in protein synthesis. The activity of eukaryotic initiation factor 4A (eIF4A) was strongly curtailed, whereas all other factors were unaffected. At a concentration of 250 .mu.M, AMP, ADP and ATP protected eIF4A against FSBA inactivation, whereas at a concentration 50 .mu.M, protection was afforded only by ATP, GTP did not pretect at a concentration of 250 .mu.M. In another approach, the substrate analog 2'',3''-O-(2,4,6-trinitrophenyl)adenosine 5''-triphosphate (TNP-ATP) inhibited protein synthesis in a manner, at least in part, competitive with ATP. Supplementing a TNP-ATP inhibited reaction with eIF4A substantially reversed the inhibition. Except for a small effect by factor C1, no reversal was obtained with any other component. Finally, a preincubation of ribosomes with ATP, [tobacco mosaic virus], mRNA and eIF4A resulted in the formation of a complex capable of TNP-ATP resistant amino acid incorporation. These data are interpreted to indicate that the primary interaction of ATP is with eIF4A. A model is proposed reconciling this conclusion with other observations relevant to the mRNA.cntdot.ribosome attachment reaction.