Abstract
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.
Highlights
That stimulates the processivity of eukaryotic translation factor 4A (eIF4A); eIF4H, a 25-kDa protein that enhances the stimulatory activity of eIF4B; eIF4E, a 25-kDa cap-binding protein; and eIF4G, a 185-kDa protein that binds most factors involved in mRNA recruitment and anchors them to the 40 S subunit through binding to eIF3
ATP hydrolysis reactions were conducted in buffer containing 100 mM KCl at pH 7.5, since it was shown [30] that eIF4A hydrolyzes ATP with at least a 3-fold higher rate at these salt and pH conditions compared with low salt and pH. cpC stimulation of poly(A)-dependent ATP hydrolysis by eIF4A was similar to that of rabbit reticulocyte eIF4F (Fig. 2A; compare lanes 5 and 8)
EIF4A was discovered as a factor involved in initiation complex formation
Summary
Materials—m7GTP-Sepharose, heparin-Sepharose CL-6B, and a Mono Q HR 5/5 column were obtained from Amersham Biosciences. Prior to performing binding or ATPase assays, purified eIF4G fragments were passed over desalting Econo-Pac® 10 DG columns to replace the buffer with buffer B100 (20 mM Tris-HCl, 100 mM KCl, 2 mM dithiothreitol, 0.1% (v/v) Tween 20, and 10% (v/v) glycerol, pH 7.5). Reactions (15 l) containing various recombinant eIF4G fragments (1 M), various forms of eIF4A (1 M), 32P-labeled RNA (0.3 M), RNasin (10 units), buffer B100, 2 mM MgCl2, 0.25 mM spermidine, and 1 mM ATP were preincubated for 10 min at 37 °C. After 40 min of preincubation of S-eIF4G fragments with eIF4A on ice, proteins were mixed with at least a 10-fold molar excess of SProtein-agarose and incubated for 1 h in buffer B150 containing 0.3 mg/ml bovine serum albumin at 4 °C. The data were fit to the Michaelis-Menten equation with iterative least squares minimizations
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