7-methylguanosine-triphosphate and 7-methyl-diguanosine-triphosphate

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.

Topics: Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Binding, Competitive; Dinucleoside Phosphates; Eukaryotic Initiation Factor-4E; Fluorescence; Molecular Sequence Data; RNA Cap Analogs; RNA Cap-Binding Proteins; RNA, Messenger; Titrimetry; Tryptophan; Tyrosine

The binding of analogues of the 7-methylguanosine-containing cap, m7GTP and m7GpppG, to eIF-4E from human erythrocytes as a function of pH, temperature, and ionic strength is described. From the pH-dependent binding of m7GTP and m7GpppG to eIF-4E, a new model describing the nature of the cap.eIF-4E interaction is proposed. The thermodynamic values and ionic strength dependence of binding are consistent with a binding site which is primarily hydrophobic. Fluorescence and circular dichroism data indicate that tryptophan residues may be involved in base-stacking interactions with the cap in a somewhat buried environment. The model presented here confirms the earlier proposal [Rhoads et al. (1983) Biochemistry 22, 6084-6088] that the enolate tautomer of the cap is preferred for interaction and further proposes that the interaction is with a protonated amino acid residue, such as histidine, while stacking with an aromatic amino acid, such as tryptophan.

Topics: Circular Dichroism; Dinucleoside Phosphates; Eukaryotic Initiation Factor-4E; Hydrogen-Ion Concentration; Peptide Initiation Factors; Protein Binding; RNA Cap Analogs; RNA Caps; RNA, Messenger; Spectrometry, Fluorescence; Temperature