Weak binding affinity of human 4EHP for mRNA cap analogs - PubMed (original) (raw)

Weak binding affinity of human 4EHP for mRNA cap analogs

Joanna Zuberek et al. RNA. 2007 May.

Abstract

Ribosome recruitment to the majority of eukaryotic mRNAs is facilitated by the interaction of the cap binding protein, eIF4E, with the mRNA 5' cap structure. eIF4E stimulates translation through its interaction with a scaffolding protein, eIF4G, which helps to recruit the ribosome. Metazoans also contain a homolog of eIF4E, termed 4EHP, which binds the cap structure, but not eIF4G, and thus cannot stimulate translation, but it instead inhibits the translation of only one known, and possibly subset mRNAs. To understand why 4EHP does not inhibit general translation, we studied the binding affinity of 4EHP for cap analogs using two methods: fluorescence titration and stopped-flow measurements. We show that 4EHP binds cap analogs m(7)GpppG and m(7)GTP with 30 and 100 lower affinity than eIF4E. Thus, 4EHP cannot compete with eIF4E for binding to the cap structure of most mRNAs.

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Figures

FIGURE 1.

FIGURE 1.

Amino acid alignment of human eIF4E (gi:4503535) with 4EHP (gi:3172339) performed with CLUSTALW (Thompson et al. 1994). Residues that are identical in proteins are shadowed in black and conserved substitutions in gray. The conserved residues that play a role in cap binding by eIF4E (Marcotrigiano et al. 1997), with changes in 4EHP, are labeled above. Stars below the lines indicate the positions of eight evolutionarily conserved tryptophan residues in eIF4E.

FIGURE 2.

FIGURE 2.

Purified on ion-exchange column proteins visualized on a Comassie-stained 15% SDS-PAGE gel. Lane 1, protein marker weight standards (Sigma); lane 2, human eIF4E; lane 3, human eIF4EW56Y; and lane 4, human 4EHP.

FIGURE 3.

FIGURE 3.

(A) Fluorescence titration curves for binding m7GTP to human eIF4E (△), its eIF4EW56Y mutant (●), human 4EHP (■), and fitting residuals. Titrations were carried out in 50 mM HEPES/KOH (pH 7.2), 0.5 mM EDTA, and 1 mM DTT adjusting to I = 150 mM by KCl at 20°C. Protein fluorescence, presented as relative value, was excited at 280 nm and observed at 337 nm. The observed increasing fluorescence signal at a higher concentration of m7GTP originates from free-cap analog emission. (B) Graphical comparison of Gibbs free energy of binding (Δ_G_o) for association of cap analogs with human eIF4E, its mutant, and human 4EHP, calculated from obtained association constants (_K_as).

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