Interaction between the NH2-terminal domain of eIF4A and the central domain of eIF4G modulates RNA-stimulated ATPase activity.

The eukaryotic translation factor 4A (eIF4A) is a member of DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples ATP hydrolysis to RNA binding and duplex separation. eIF4A participates in the initiation of translation by unwinding secondary structure in the 5'-untranslated region of mRNAs and facilitating scanning by the 40 S ribosomal subunit for the initiation codon. eIF4A alone has only weak ATPase and helicase activities, but these are stimulated by eIF4G, eIF4B, and eIF4H. eIF4G has two eIF4A-binding sites, one in the central domain (cp(C3)) and one in the COOH-terminal domain (cp(C2)). In the current work, we demonstrate that these two eIF4G domains have different effects on the RNA-stimulated ATPase activity of eIF4A. cp(C3) stimulates ATP-hydrolytic efficiency by about 40-fold through two mechanisms: lowering K(m)(RNA) by 10-fold and raising k(cat) by 4-fold. cp(C3) also stimulates RNA cross-linking to eIF4A in an ATP-independent manner. Studies with eIF4G and eIF4A variants suggest a model by which cp(C3) alters the conformation of the catalytic site to favor RNA binding. cp(C2) does not stimulate ATPase activity and furthermore increases both K(m)(ATP) (at saturating RNA concentrations) and K(m)(RNA) (at subsaturating ATP concentrations). Both cp(C3) and cp(C2) directly interact with the NH(2)-terminal domain of eIF4A, which possesses conserved ATP- and oligonucleotide-binding motifs, but not with the COOH-terminal domain.

Mass spectrometric analysis of the N terminus of translational initiation factor eIF4G-1 reveals novel isoforms.

In eukaryotes, translation initiation factor 4G (eIF4G) acts as the central binding protein for an unusually large number of proteins involved in mRNA metabolism. Several gene products homologous to eIF4G have been described, the most studied being eIF4G-1. By its association with other initiation factors, eIF4G-1 effects mRNA cap and poly(A) recognition, unwinding of secondary structure, and binding to the 43S initiation complex. Multiple electrophoretic isoforms of eIF4G-1 are observed, and multiple cDNAs have been reported, yet the relationship between the two is not known. We report here a new cDNA for eIF4G-1, present as a previously unidentified human expressed sequence tag, that extends the long open reading frame, provides a new in-frame initiation codon, and predicts a longer form of eIF4G-1 than reported previously. eIF4G isoforms from human K562 cells were cleaved with recombinant Coxsackievirus 2A protease and the N- terminal domains purified by m(7)GTP-Sepharose chromatography and polyacrylamide gel electrophoresis. Proteins were digested with proteolytic enzymes and peptides masses determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry. In selected cases, peptides were sequenced by electrospray-mass spectrometry fragmentation. This identified the N termini of the three most abundant eIF4G-1 isoforms, two of which had not previously been proposed. These proteins appear to have been initiated from three different AUG codons.