Section 23.2.4.7. The recognition of alkylated bases

The complex of VP39 with capped RNA also illustrates a final example of the diversity of protein–ligand interactions in the specific recognition of the 7-methylguanosine cap. When guanosine is methylated at the N7 position, a positive charge is introduced to the π-ring system of the base. Eukaryotic cells utilize the methylation of a guanosine base at the N7 position as a tag or cap for the 5′ end of messenger RNA. The m⁷G5′ppp mRNA cap is specifically recognized in the splicing of the first intron in nascent transcripts, in the transport of mRNA through the nuclear envelope and in the translation of the message by the ribosome (Varani, 1997). Two structures of specific m⁷G binding proteins are now known: VP39 and the ribosomal cap-binding protein IF-4E, (Hodel et al., 1997; Marcotrigiano et al., 1997). Each structure offers clues as to how the proteins can discriminate between the charged methylated m⁷G base and the unmodified guanosine base. The m⁷G base is stacked between aromatic protein side chains and hydrogen bonded to acidic protein residues (Fig. 23.2.4.5). One long-held hypothesis is that IF-4E, with dual tryptophan residues, binds specifically to the positively charged form of the base through a charge-transfer complex (Ueda, Iyo, Doi, Inoue & Ishida, 1991). The formation of a charge-transfer complex is evident in small-molecule studies and spectroscopic studies with IF-4E (Ueda, Iyo, Doi, Inoue, Ishida et al., 1991). However, VP39 performs the same discrimination with the much less electronegative phenylalanine and tyrosine side chains (Hodel et al., 1997). So far, no charge-transfer complex has been observed in VP39.

Figure 23.2.4.5| top | pdf | The specific recognition of the messenger RNA 7-methylguanosine cap. (a) The residues contacting the m7G base in the cap-binding protein, IF-4E (Marcotrigiano et al., 1997). (b) The residues interacting with the cap in the vaccinia RNA methyltransferase VP39 (Hodel et al., 1997). Both proteins bind to the charged, methylated base by stacking aromatic amino acids on both sides of the base.

The recognition of charged methylated bases is important not only in mRNA processing, but also in the repair and recognition of DNA damaged by alkylating carcinogens. The mechanism by which the charged m⁷G base is recognized is probably similar to how other positively charged bases, such as 3-methyladenosine, O2-methylcytosine and O2-methylthymidine, are recognized. In fact, the E. coli DNA repair enzyme, AlkA, will catalyse the glycolysis of all of these bases (Lindahl, 1982). The structure of AlkA is known, but only in the absence of a substrate (Labahn et al., 1996). In this structure, a number of solvent-exposed tryptophan residues are found at the putative active site. This observation suggests that AlkA may recognize positively charged bases through an aromatic `sandwich', much like that found in IF-4E and VP39.