Structural Requirements for the Specific Recognition of an m7G mRNA Cap

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Abstract
7-Methylguanosine (m7G), also known as the mRNA “cap”, is used as a molecular tag in eukaryotic cells to mark the 5‘ end of messenger RNAs. The mRNA cap is required for several key events in gene expression in which the m7G moiety is specifically recognized by cellular proteins. The configurations of the m7G-binding pockets of a cellular (eIF4E) and a viral (VP39) cap-binding protein have been determined by X-ray crystallography. The binding energy has been hypothesized to result from a π−π stacking interaction between aromatic residues sandwiching the m7G base in addition to hydrogen bonds between the base and acidic protein side chains. To further understand the structural requirements for the specific recognition of an m7G mRNA cap, we determined the effects of amino acid substitutions in eIF4E and VP39 cap-binding sites on their affinity for m7GDP. The requirements for residues suggested to π−π stack and hydrogen bond with the m7G base were examined in each protein by measuring their affinities for m7GDP by fluorimetry. The results suggest that both eIF4E and VP39 require a complicated pattern of both orientation and identity of the stacking aromatic residues to permit the selective binding of m7GDP.