Methyl esterification of m7G5′p reversibly blocks its activity as an analog of eukaryotic mRNA 5′-caps

Abstract The methyl ester of m 7 G 5′ p was synthesized by a carbodiimide-catalyzed reaction of G 5′ p with methanol followed by dimethylsulfate alkylation. Comparative spectral analyses indicated that m 7 Gp · methyl ester retained the rigid conformation characteristic of the messenger RNA cap analog, m 7 G 5′ p but not its strong inhibitory activity against initiation of capped mRNA translation. Attachment of reovirus mRNA to wheat germ ribosomes, crosslinking of capbinding protein to the 5′-end of oxidized mRNA, and stimulation by this protein of capped mRNA translation in HeLa cell extract were all several-fold more sensitive to inhibition by m 7 G 5′ p than to m 7 Gp · methyl ester. Conversion of the esterified analog to m 7 G 5′ p by digestion with venom phosphodiesterase restored completely the ability to inhibit initiation complex formation. The results indicate that structural features of the 5′-terminal m 7 G cap of mRNA over and above preferred conformation are recognized during eukaryotic protein synthesis.